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ProteOn™ XPR36 Experimental Design and Application - Bio-Rad

1. CBS Benzenesulfonamide Furosemide 200 z z 2 g g 100 E C E 5 S amp 504 3 2 Q cc ao oO O 20 T T T 20 T T T T 50 T T T T T T 100 O 100 200 300 400 100 50 O 50 100 150 200 50 O 50 100 150 200 250 300 Time sec Time sec Time sec 7 fluoro 2 1 Dansylamide 3 benzoxadiazole 4 sulfonamide Methylsulfonamide 100 2 R 78 2 g 564 Do PARIA 5 5 a aol A 8 g H 3 i Wet My oc ag 12 4 Oo 0 a T 10 T 10 T T 25 20 65 110 155 200 60 12 204 336 468 600 50 O 50 100 150 200 Time sec Time sec Time sec Acetazolamide Sulfanilamide 1 3 benzenedisulfonamide 120 ima T T ae 5 5 5 O Q Q g ce or a 12 T T ie T T T 20 T T T T 70 28 126 224 322 420 34 16 66 116 166 216 50 O 50 100 150 200 250 Time sec Time sec Time sec Sulpiride 164 1147 A 5 64 P rc 144 36 l i i 48 2 52 102 152 Time sec Fig 3 10 Screening of sulfonamide inhibitors to carbonic anhydrase isozyme RU response units Table 3 1 Results of the interactions of CAII MW 29 000 with ten different inhibitors Highest Concentration ka Ky Ko has Analyte MW Used uM M sec7 sec M RU Sulpiride 341 250 2 52 x 108 0 26 PONTON 188 Sulfanilamide 172 50 2 40 x 104 0 12 4 8 x 1078 112 Furosemide 331 50 5 15 x 104 0 04 fl x O 180 CBS 201 50 2 83 x 104 0 03 1 2 x 1078 105 Dansylamide 250 10 1 33 x 10 0 09 6 5 x 107 105 1 3 benzene 236 10 1 11 x 108 0 09 8 1 x 10 7 9
2. i a i sam rd dyert iaia a iee ror 2 os sre a Puna Type l Fra In the menu bar click Process select Double Reference and 2nd reference choose the appropriate blank buffer reference namely Double ea re Reference in the submenu _ EEN i soas Cee atistin gt gt Sead Sper S rero a E S N Open the Interaction screen and deselect the unused or outlier oO Interaction rae T seai interaction spots to be excluded from sensorgram fitting Click ja Apply and then Close Y See O l Q Open the Create Dataset screen type a dataset name in a Create dataset the dialog box and click Create to create a dataset with the processed sensorgrams NO Good sensorgrams Troubleshooting with the data processing steps 84 Experimental Design Data Analysis Processed data Choose dataset What type In the Navigator sidebar select the Analysis Dataset tab and choose a dataset to fit 5 os mirr ir l E of analysis Q D Select type 2 O D c N O O pa a 2 y dp Choose model KINETICS EQUILIBRIUM Select Analysis and choose Equilibrium in the submenus Select Analysis and choose Kinetic in the submenus Select type eee Or T L Mt O Choose the appropriate kinetic fitting model in the Choose Model box If th
3. 3 3 4 Nucleic Acid Interaction Analysis The interactions between proteins and nucleic acids have increasingly drawn attention in the biological research community as the understanding of these interactions often bridges genomics and proteomics The ProteOn XPR36 system is capable of analyzing protein nucleic acid interactions with high performance Published Applications A Wide Range of Applications for the NLC Sensor Chip Cohen S et al 2006 Applications of the ProteOn NLC sensor chip antibody antigen DNA protein protein protein interaction analysis Bio Rad Bulletin 5449 This technical note Cohen et al 2006 describes the specificity and stability of biotinylated ligands captured to the ProteOn NLC sensor chip Three common biomolecular interaction models representing antigen antibody DNA protein and protein protein interactions were analyzed using the One shot Kinetics approach These three interaction types were analyzed in high throughput with the 6 x 6 interaction array Figure 3 26 Trp repressor and trp operator Stephen A et al 2009 Determining the binding kinetics of HIV 1 nucleocapsid protein to six densities of oligonucleotide using the ProteOn XPR36 protein interaction array system Bio Rad Bulletin 5846 ProteOn XPR36 system is used to analyze the binding kinetics of the HIV 1 nucleocapsid protein NC with a short deoxynucleotide d TG Stephen et al 2009 Six different surface densities of d TG
4. k and k a d Equilibrium S Association Dissociation Response RU Baseline Regeneration Time sec id a Published Applications Characterization of the VWF A1 Domain Epitope for an Antibody Kulman J 2010 A calcium dependent immunocapture strategy for enhanced throughput SPR Bio Rad ProteOn Webinar Series In order to resolve the binding epitope of VWF von Willebrand factor A1 domain against a monoclonal Ab of interest the interaction was analyzed using the ProteOn XPR36 system Kulman 2010 A series of At mutants were designed and expressed in cell lines and were screened against the antibody using the One shot Kinetics approach The measurement was taken over a temperature range to achieve thermodynamic analysis Figure 3 23 From this experiment both kinetic constants and transition state thermodynamic parameters were obtained According to the values of free energy enthalpy and entropy changes in the interactions the contribution of different residues at the potential epitope region was resolved Compared to conventional biosensors the high throughput of the ProteOn XPR36 system shortens the experiment time from a month to days Sr N633A Run 1 Run 2 Run 3 Run 4 Run 5 ial DD a gt Perrier gt gt PEPPE a a al I Fig 3 23 Thermodynamic analysis for epitope mapping in the Ag Ab interaction 46 Applications 3 3 3 Histidine Tag
5. m a j 2 Channel Reference gt p a ie fe R amp Pae x PERTE Double Reference gt Row AL Interspot referencing is unique to the ProteOn XPR36 Instrumei Buto Process mail f j system Instead of consuming potential interaction Protoco 4 Injection Alignment gt Injection AZ f Run 44 Baseline Alignment gt Salah triachion Aa surfaces this reference method employs the interval Data Artifact Removal gt AS surfaces adjacent to interaction surfaces Compared eid Manual Alignment VY A with the traditional channel reference the interspot reference has the advantages of immediate proximity Panel Type A to interaction spots and the conservation of interaction spots The immediate proximity enhances the fes ao l pm 3 Remove Processing Fig 4 7 ProteOn Manager software presents all the referencing referencing quality options available in the ProteOn XPR36 system for selection Blank surface referencing also known as channel referencing is performed on a blank surface either an empty or an irrelevant protein coated surface with an analyte solution flowing over it The reference responses are collected on blank surfaces during the analyte injection that is blank surface reference blank surface analyte solution as shown in Figure 4 8 Blank surface referencing is used to correct for bulk effect and NSB 70 Experimental Design Ligand channels Raw Data Analyte Inje
6. For ease of use ProteOn Manager software allows the user to conduct all the sensorgram processing steps with a single command Selecting Auto Process in the Process menu sequentially performs injection alignment baseline alignment and artifact removal RE One Shot Kinetics Experiment ProteOn Manager File Edit DA 28 Help rb it RQ 0 view Process Analysis Tools Channel Reference adl Double Reference gt l etics Experiment Instrumet AS Auto Process Protoco 4 Injection Alignment gt Run Baseline Alignment gt i nanan Data HH Artifact Removal gt Auto amp Manual Alignment Selected Remove Processing Panel Type Fig 4 6 The automatic artifact removal function offered in ProteOn Manager software 69 ProteOn XPR36 Experimental Design and Application Guide 4 4 4 Sensorg ram Referencing Ligand Surface Analyte Injection Sensorgram referencing is the most important step in eee p data processing The subtraction of references removes ienee espe artifacts of refractive index change bulk effect from the analyte sample nonspecific binding NSB of the analyte and impurities on the sensor chip surface and changes of the ligand surface There are two types of referencing in SPR analysis blank surface referencing and blank buffer referencing A blank surface reference is used to correct for bulk effect and NSB and a blank buffer reference is us
7. In drug discovery research isoaffinity and screening graphs are frequently used to view the screening results for positive hit pickup ProteOn Manager software can be used to create either graph In the Analysis menu choose Isoaffinity Graph or Screening Graph to display the graph Both graphs allow the selection of target datasets in the left panel Examples of an isoaffinity graph and a screening graph are presented in Figure 4 21 Experimental Design HE One Shot Kinetics Experiment Analyte Step 17 Chl Row Kinetic Langmuir Pr Select points to be displayed ia the graph y rae Aa E File Edit View Process Analysis Tools Help D a J view Quick Start x Navigator show Interspot Data etics Experiment Ir a Show Point Minimized Data Show Smoothed Data ka 6 19E 05 i Ms kd 9 50E 05 7 NOW base ne Alig eg Va Show Range Selection Anal J Show Markers GQ Analyte St Show Report Point Markers 400 600 800 Gi Analyte st JY Show Kinetic Results Time s Kinetic Show All Rav BQ Analyte St i ka 6 42E 05 1 Ms kd 9 78E 05 l E Kinetic YH Select All Graphs Ctrl La asia sad igre Lp Manual Zoom E Ligand Ste R Zoom Out Screening Graph Time s Normalized Screening Channels Average Threshold Titles Chart Normalized Screening Fit Fit line thickness 1pt w se same color as series Normalized Screening Screening Moteculsr Weight 10
8. 6 L ge Interaction at intersection 2 823 RU ligand density 2 053 RU ligand density 160 S 160 140 _ A RAinig a giaren iaka 140 1204 IA eee A epii 120 100 4 7 100 2 80 TB ao O O DBO piirirepirjiei dis ea l 2 60 ree c 40 4 T 40 ii dapa 20 g f j Fa P aiaiai A A a aa a a edited aaa a aia AAA 20 o er 0 aL a a as O 2 4 6 8 10 12 Time min Time min 1 702 RU ligand density 1 468 RU ligand density 160 160 140 140 120 120 2D j hii T 1004 aA kiddih aaia da E i00 D 80 4 EEEE EONTR DA a a an Door EE D 80 GON a peaa nL ih ater ile chon einai sr rarei ei O O P 60 ca aa iad 60 e ify T 404 fff pemenang nS er 0 E eee ences gree OETAN 204 IZ aeee woes j Fa iiaiai aiia i l iY a OA A ee th net aa ana nann aaan aaan aa N N O E E OE EN SNO N 0 Ww o nen I O 2 4 6 8 10 12 O 2 4 6 8 10 12 Time min Time min 1 368 RU ligand density Reference channel 100 8 6 i Salen ae A Dae aw De Rae gt al E UEAN D LAE AAA 4 a j am 2 oO i oO 807 ff RTE tte D 5 Wy O AAA nab meetin AAA BNE A AA n RAABAN AASA A aa A Q Q EF popes g Ser cc F cc f 4 J 6 4 8 O 2 4 6 8 10 12 O 2 4 6 8 10 12 Time min Time min Fig 3 2 One shot Kinetics approach for the kinetic analysis of the IL 2 cytokine IL 2 antibody interaction
9. 7 XLSX File i sensorgram set under the dataset If the Unsaved oles processed data will be lost dialog box appears Text File Y select Yes Choose Print in the submenu ___ maeme fzo o 200 400 600 800 1000 1200 1400 Time s a T a Option 2 Copy Sensorgrams to a ProteOn XNPRI6 Kinetic Report Kinetic Langer SOW s arem eeatt a Presentation Siep 17 EL LI Aah RE Ah Aen pil 48 L at th dentine gl OS Emer CERTE T opeen pp yan 1 In the Navigator sidebar enter the Analysis Dataset menpe tab and choose a dataset When a graph with overlaid fitting curves is preferred select an analyzed sensorgram set under the dataset 2 Click to select the graph to be copied Hold down the Ctrl key to select multiple graphs or press Ctrl A to select all graphs 3 Right click any selected graph and choose Copy A Graph A status bar is shown When the copy process is completed paste the graph into the presentation 2 In the Report Options dialog box select the items to be included in the report and then click OK Option 3 Export Data to a Spreadsheet The experiment report is generated in the Report 1 In the Navigator sidebar enter the Analysis Dataset Preview screen tab and choose a dataset If the curve fitting data are needed select an analyzed sensorgram set under the dataset PreteOu XPR36 Kietie Report Kietie Langmuir cx 2 Click to select the graph to be copied Hold down the Jee a Laat she
10. Fig 4 28 Choosing reference and EVC injections and viewing the R value of the fit 5 Double click the thumbnail plots with low R values and then click on the bad data point to remove it The excluded data point is represented by an empty circle and will not be included in the analysis At least three solid data points must be selected from each calibration plot Figure 4 29 RU ligand RU reference 400 200 O 200 400 RU reference RU ligand RU reference 400 200 O RU reference 200 Fig 4 29 Viewing the quality of the data for calibration A original data showing all five data points included in the calibration plot B modified calibration plot after removal of a data point 82 Experimental Design 6 In the second wizard step select all the analyte steps for which you want to apply the EV correction Click Finish to apply the reference and display the corrected data Figure 4 30 10 Ke 22 wo w sae 400 ae at 400 T Page ale lt Dek Fosh Carcel Fig 4 30 Choosing the steps for applying EV calibration 7 Apply a double reference if desired 8 Autoprocess the data 9 Save the processed dataset using the Create dataset option 83 ProteOn XPR36 Experimental Design and Application Guide 4 1 Data Processing and Analysis Flowchart Raw data o In the menu bar clic
11. G 5 D or 40 O 40 80 Time sec Fig 3 25 ProteOn HTG and HTE sensor chips A sensorgrams of the interaction between the histidine tagged protein A and IgG showing the ability of the HTG chip to resolve high affinity kinetics requiring long dissociation times Protein A was captured to approximately 60 RU and human IgG was injected in a twofold dilution series ranging from 100 6 3 nM B sensorgrams of the interaction between histidine tagged Erk2 a MAP kinase and the inhibitor Purvalanol B 432 9 Da showing that small molecules can be screened using the HTE chip Erk2 was captured to approximately 12 800 RU and Purvalanol B was injected in a threefold dilution series ranging from 50 uM to 0 62 uM RU response units 47 ProteOn XPR36 Experimental Design and Application Guide Characterization of the Interaction of ODC and Az Cohavi O et al 2009 Docking of antizyme to ornithine decarboxylase and antizyme inhibitor using experimental mutant and double mutant cycle data J Mol Biol 390 503 515 This article Cohavi et al 2009 uses the ProteOn XPR36 system to analyze the interaction between ornithine decarboxylase ODC and a regulatory protein antizyme Az The binding sites of ODC on Az were mapped using high throughput mutagenesis and computational docking Double mutant cycle DMC analysis between residues on Az and ODC was used to obtain further insights on the structure and function of the complexes
12. RU response units 3 4 Biological Assays Due to the vast array of applications for SPR label free interaction protocols for the ProteOn XPR36 must be tailored to specific experimental needs to obtain high quality SPR data With the deposition of six or more ligands on one sensor chip the ProteOn XPR36 system s unique 6 x 6 interaction array allows for flexibility in the design of biomolecular interaction assays streamlining assay design Interrogating these different ligands with six separate analytes facilitates experimental optimization by enabling the real time detection of up to 36 different biomolecular interaction events simultaneously 3 4 1 Assay Design and Optimization Assay design is essential for obtaining high quality SPR results Selecting and optimizing the most suitable experimental conditions is of utmost importance for assay accuracy and reproducibility The novel 6 x 6 interaction array of the ProteOn XPR36 system provides the versatility for many different types of experiments on a single platform at high throughput This advantage allows for simultaneous investigation of multiple experiment conditions of an interaction and also rapid reproducibility testing Because of the unique advantages in assay design the ProteOn XPR36 system provides the highest efficiency and accuracy compared to other SPR platforms available Advantages include Rapid screening for reagents Flexible assay configuration Mul
13. San Diego Dec 2011 The histidine tag is one of the most widely used tags in protein purification Recombinant proteins containing the histidine tag are easily captured by a tris nitrilotriacetate tris NTA surface Bio Rad offers the HTG and HTE chips for capturing histidine tagged proteins for interaction analysis The chips employ a novel tris NTA surface chemistry which provides higher stability and selectivity ATP Response RU T T T T T T 80 40 O 40 80 120 160 Time sec Purvalanol B T T T T T T T 80 40 O 40 80 120 160 Time sec JAK3 Inhibitor VI 2 40 pe Z f N 3 20 A 0 M T T T T T T T 80 40 O 40 80 120 160 Time sec Response RU Response RU Staurosporin T T T T T T T 80 40 O 40 80 120 160 Time sec Amino Purvalanol A 804 40 O T T T T T T T 80 40 O 40 80 120 160 Time sec 1 Naphthyl PP1 40 20 10 T T T T T T T 80 40 O 40 80 120 160 Time sec in capturing histidine tagged proteins compared to the traditional NTA surface chemistry and allows for surface regeneration This surface intercalates at three points with a histidine tagged protein rather than a single point for the NTA surface The HTG chip is ideal for the analysis of protein protein and protein peptide interactions and HTE chip for protein small molecule interactions In this work Luo et al 2011 small molecules bi
14. Shown are the six sets of six sensorgrams generated in a single analyte injection step Each set of six sensorgrams displays the responses from the six IL 2 cytokine concentrations 80 nM 40 nM 20 nM O nM 5 nM 2 5 nM interacting with one immobilization level of IL 2 antibody Sensorgrams are shown for the five levels of IL 2 antibody immobilization ligand density and the reference channel Black lines represent the global fit of the sensorgrams to a 1 1 kinetic interaction model RU response units 31 ProteOn XPR36 Experimental Design and Application Guide Yousef M 2007 Advances in rapid monoclonal antibody screening Am Biotech Lab 25 26 28 The article Yousef 2007 describes an alternative method for the rapid screening of monoclonal antibodies using multiplexed SPR and the One shot Kinetics approach of the ProteOn XPR36 system The ProteOn XPR36 system was used to screen supernatants to identify high affinity mAb candidates against human IL 12 and hemoglobin E Over 250 supernatants were screened in 12 5 hr in one experiment using a single sensor chip There was no need to purify antibodies from the supernatants prior to analysis 36 Ligand Array for Antibody Kinetic Screening and Epitope Binding Abdiche YN et al 2011 Expanding the ProteOn XPR36 biosensor into a 36 ligand array expedites protein interaction analysis Anal Biochem 411 189 151 Lindquist K 2011 Enhancing th
15. The articles illustrate a promising workflow for the rapid large scale production of vaccines Vaccine Potency Determination Khurana S et al 2014 Novel antibody independent receptor binding SPR based assay for rapid measurement of influenza vaccine potency Vaccine 32 2188 2197 An antibody independent simple high throughput receptor binding SPR based potency assay is proposed using the ProteOn XPR36 system The assay measures the binding between influenza vaccine strains in sample flow and synthetic glycans immobilized on the surface of a chip The active forms of hemagglutinin in vaccine samples are quantified by the initial binding slopes and thus vaccine potency is determined The advantages of this SPR based potency assay are 1 it does not require any reference antiserum sample and 2 it can be used for rapid hemagglutinin quantitation and vaccine release 56 Applications The ProteOn XPR36 system shows high performance in the potency assay and allows the testing of multivalent vaccines Excellent concordance is shown between the SPR based potency assay and the standard single radial immunodiffusion SRID assay Figure 3 37 3 5 2 Clinical Diagnostics The compatibility with clinical samples extends the applications of the ProteOn system to clinical diagnostics It has been used as an efficient tool in active serum component quantitation Published Applications Antibody Drug Companion Diagnostics Thoren K
16. and running steps for an experiment Note The ProteOn Manager software also indicates in the Maintenance Template means a saved protocol intended for reuse and Experiment Status table whether other maintenance protocols are required at this means an implemented protocol You are able to create a new protocol time from all these file types 114 Quick Guides 2 Shut down the instrument Skip this step if the instrument is in continuous use A Inthe Navigator panel go to the Instrument tab and select Instrument Control B Click Shutdown and select either Immediate Shutdown or Long Term Shutdown An MNT chip in the instrument is required for long term shutdown Follow the pop up instruction to load the reagents and click Next to start the shutdown process C Wait until the shutdown process is completed Press the power button on the left side of the instrument to the O position to turn off the instrument Note If the instrument is in continuous use it is recommended to keep the system in distilled water during the idle time for example overnight or over the weekend 7 2 4 Import Export Experiment Files 1 Export experiment files A In the menu bar click File select Export and choose Experiment Protocol File in the submenu to open the database browser Select the experiment file to export Hold the Ctrl key to select multiple experiment files B Click Export and select the target folder to store the exporte
17. association of the analyte to the ligand Mass transport 4 2 8 Summary The protocols and methods provided in this chapter are meant as a foundation to create your own experimental protocols and methods specific to your individual research projects Exact optimal experimental conditions will vary according to the specific application We strongly recommend the conditions be optimized and determined as this will lead to consistent and high quality SPR results Try acapture method or biotinylation of the acidic protein Increase contact time Lower flow rate Increase protein concentration Optimize pH Use positive control to gauge the activity of the immobilized protein Try a capture method to ensure correct orientation Immobilize in presence of protecting molecule or cofactor Reduce ligand density or increase analyte flow rate 65 ProteOn XPR36 Experimental Design and Application Guide 4 3 Guide to Analyte Injection on the ProteOn XPR36 System 4 3 1 Introduction A simple binding interaction analysis by SPR starts with the immobilization of ligand to the sensor chip surface as described in section 4 2 This is followed by the addition of the analyte of interest to the buffer flowing over the ligand surface The interaction of the ligand and analyte is measured by the SPR instrument as a change in refractive index over time From this the association ka or Kon dissociation ky or Korr and equilibrium Kp
18. k k The parameters are obtained from the data fitting of the association equilibrium optional and dissociation phases of a Sensorgram e ee cee o Baseline Association Equilibrium Dissociation Regeneration Equilibrium Association Dissociation Response RU Baseline Regeneration Time sec Fig 1 1 SPR sensorgram Surface ligand Y analyte RU response units 10 ProteOn XPR36 Technology The ProteOn XPR36 system offers a distinct advantage over other SPR biosensor platforms because the unique 6 x 6 interaction array of the ProteOn sensor chips enables the One shot Kinetics approach measuring the interaction of one ligand with a six concentration series of one analyte in a single injection This approach eliminates the need for traditional regeneration of the sensor chip between analyte injections which often deteriorates the ligand surface Using enhanced microfluidic delivery and XPR technology the ProteOn XPR36 system can immobilize up to six separate ligands on a single sensor chip in six separate flow cells and then rotate the sensor chip 90 degrees to flow up to six separate analytes over the ligand surfaces Figure 1 2 This unique feature of the ProteOn allows for the detection of up to 36 separate interactions on a single sensor chip and significantly increases the throughput of SPR biosensing In a recent study the ProteOn XPR36 system was used to immobi
19. or liposomes The NLC chip comes prepared with NeutrAvidin immobilized to its surface This chip is suitable for subsequent protein protein and protein nucleic acid interaction analysis 64 Experimental Design The HTG and HTE chips feature a novel tris NTA 3 x NTA surface for stable capture of histidine tagged proteins The HTG chip has a compact capacity for protein protein interaction analysis and the HTE chip has a high capacity for protein small molecule interaction analysis Refer to Chapter 5 section 5 2 for the ligand capture conditions for NLC HTG and HTE chips Table 4 1 Troubleshooting Problem Possible Causes Solution Working with acidic proteins Acidic proteins are difficult to immobilize by amine coupling as they require buffer conditions that may be denaturing and may neutralize the activated negative sulfo groups on the chip surface and prevent attraction Enhance immobilization The amount of protein immobilized is too low Ligand immobilized but no interaction Protein may no longer be active because the immobilization conditions are too harsh too strong OH or salts The active site on the protein may be buried because of the random immobilization orientation Enzymes may be active only if immobilized in the presence of another molecule or cofactor or to protect the active binding site This occurs when the rate of diffusion of the analyte from the flow is slower than the rate of
20. ra ual Channel Reference Real Time Double Reference Fig 6 5 SPR referencing options provided by the ProteOn XPR36 system The ProteOn XPR36 system offers two channel A __Interspot referencing i a i i referencing options interspot referencing the novel U eee on E Does not require a ligand channel all 36 spots are available referencing mechanism utilizing the blank surfaces for interaction analysis between interaction spots and Crain referencing casas esas the traditional SPR referencing mechanism that uses a l l l l l l l l l l l l dedicated blank channel The innovative fluidics design Fi of the ProteOn XPR36 system also offers two double referencing options injection referencing the traditional referencing mechanism that uses a blank running 8 l i Interspots buffer injection prior to analyte injections and real time S S j double referencing a blank real time running buffer lt lt j injection performed in parallel with analyte injections The ProteOn XPR36 system is the only SPR biosensor to feature real time double referencing that runs simultaneously with the ligand analyte interactions B Real time double referencing Provides higher data quality especially important when using Interspot and real time double referencing are unique aca uiecunccs to the ProteOn XPR386 system and their advantages are listed in Figure 6 6 Ligands Jeeudd TITTEE Analytes Blank analyte c
21. were achieved in the six ligand channels of a sensor chip and five concentrations of NC were tested in the analyte channels The low ligand density channels showed excellent sensorgrams Figure 3 27 The results are consistent with previous work A Channel 1 B Channel 2 100 D a a N N Cc O O Q Q ep N od od cc cc Time sec Channel 3 0 200 400 600 800 1 000 O 200 400 600 800 1 000 Time sec Channel 4 60 pili ont T m i 40 A T Akiba bednetigt T A ppan trin a VA O Lyf P TRE 8 20 ATT aian LL p d i A 7 dusk treats tt led a o hata aa Lit ie O i 2 Time min Fig 3 26 Interaction analysis of an oligonucleotide containing the trp operator sequence and the trp repressor protein Tro repressor concentrations 8 nM 6 nM 4 nM 2 nM 1 nM and 0 5 nM RU response units Response RU Response RU nen ia 5 pz T T a O 200 400 600 800 1 000 O 200 400 600 800 1 000 Time sec Time sec E Channel 5 F Channel 6 T T T O O 200 400 600 800 1 000 O 200 400 600 800 1 000 Time sec Time sec Fig 3 27 Interaction kinetics of NC binding to different densities of d TG are compared to the Langmuir 1 1 model The black trace represents the global fit of the sensorgrams to the 1 1 interaction model The interactio
22. 7 5 6 1 9 and 0 6 UM RU response units 39 ProteOn XPR36 Experimental Design and Application Guide Fragment Screening Dolezal O 2013 Lead discovery Screening and characterization using multiplexed SPR Bio Rad ProteOn Webinar Series Peat TS et al 2012 Small molecule inhibitors of the LEDGF site of Human Immunodeficiency Virus Type 1 Integrase identified by fragment screening and structure based design PLoS ONE 7 e40147 Popplewell J 2013 SPR based fragment screening and small molecule affinity analysis using ProteOn XPR36 system BioRadiations Sep 2013 Fragment based lead discovery or fragment screening is amethod used for finding lead compounds in drug discovery It is based on identifying small chemical fragments which may bind weakly to the biological target and deriving or combining them to build a lead compound with higher binding affinity An SPR biosensor is one of the major tools used in fragment screening These articles and the webinar report different fragment screening experiments performed with the ProteOn XPR36 system Figure 3 15 Response RU 20 0 20 40 60 80 100 120 140 160 180 200 Time sec Fig 3 15 Screening of 260 360 Dalton small molecules for fragment based lead discovery RU response units 3 3 Biomolecule Characterization Unlike endpoint assays such as ELISA that measure a binding response after reaching equilibrium SPR allows for the detection of bindi
23. AVTE DRAN or 15 bas dy or be ipa tie AA Gannon UCOUN Ow OT TEND aani OWEN IY 10 T T T T T O 1 2 3 4 5 Time min Clone 3 110 T 704 i g S p gh naa aoa ids internet ead ty ANBS a N sy tly Le he Yet ahs 2 TR ah a REP UA PAR PANA COTY a ot a eee ee are Ae apd apritragt Panel breed p Alh AD 10 l O 1 2 3 4 5 Time min Fig 3 6 Screening of antibody supernatants RU response units 3 2 3 Quantikinetics SPR technology is able to determine the concentration of an analyte based on a set of standard samples of known concentrations Typically a ligand is immobilized on the sensor chip at relatively high density and analyte samples are injected The initial binding rate of the analyte is measured and correlated directly with analyte concentration The concentration of an unknown sample is calculated by comparing the binding response under these conditions to a standard curve of binding responses for known concentrations The parallel fluidics and reproducibility of the ProteOn XPR36 system enable reliable and high throughput concentration analysis of biological samples for both research applications and manufacturing and quality control processes There are two major advantages of SPR over other labeled techniques such as ELISA for concentration analysis 1 label free SPR eliminates the effort involved in labeling the analyte of interest and 2 using an SPR sensor chip with binding specificity either by using a captu
24. B e A2 B lt A2B d1 d2 Equation 6 Heterogeneous Ligand Model A heterogeneous ligand model assumes that there are two sites on the ligand that bind analyte This can occur if ligand binds to the sensor chip in different orientations resulting in different binding faces being presented to the analyte Polyclonal antibodies recognize different epitopes on the same antigen and thus would be considered a heterogeneous ligand The following equation describes binding of analyte to a heterogeneous ligand k k al a2 A B1 a B2 gt AB2 d1 d2 Equation 7 where B1 and B2 are the two separate binding sites on the ligand and A is the analyte Note that there are two separate sets of association and dissociation rate constants k k and k k to describe each binding event The binding response of a sensorgram from a heterogeneous ligand then is the sum of the binding response of two separate binding events Two State Conformation Model The two state conformation model accounts for the existence of two conformations of the bound complex This can happen if binding of the analyte to ligand triggers a change in conformation of the bound complex Equation 8 describes the two state confirmation binding model Kar Kao A B gt AB lt AB Kj d2 Equation 8 19 ProteOn XPR36 Experimental Design and Application Guide In Equation 8 AB is the first conformation of the bound complex and AB is the second conformatio
25. EDC and 100 mM sulfo NHS which are stored at 20 C until needed It is important to make this mixture fresh every time as it has a half life of 30 60 min and should therefore be used immediately After thawing and mixing you may dilute the equivolume activation solution prior to use depending on which application you are working with 62 Experimental Design Chemistry After addition of EDC and sulfo NHS to the chip the carboxyl groups react and become sulfo NHS esters During the ligand injection step the ligand preferentially binds to the esters and is amine coupled to the chip surface 4 2 3 Immobilization Many factors affect ligand immobilization including chip type level of surface activation ligand concentration size and injection parameters such as contact time injection flow rate and electrostatic attraction of the ligand to the surface Electrostatic attraction is one of the most important factors because if the ligand is not attracted to the surface there will be very little immobilization Optimizing Immobilization Conditions After amine coupling the sensor chip surface will have an overall negative charge the ligand therefore must have an overall positive charge This is achieved by determining the optimal immobilization buffer Since the ProteOn XPR36 system has six ligand channels you can easily test multiple immobilization conditions immobilization buffers of different pH to determine which gi
26. NeutrAvidin modified self assembled monolayer on the LCP sensor chip B workflow for liposome capture using the LCP chip and the LCP capturing reagent kit Chol dsDNA 1 and single stranded biotinylated DNA molecules biotin ssDNA contain complementary DNA sequences The LCP sensor chip surface is saturated with biotin ssDNA and then liposomes incubated with chol dsDNA 1 are captured to the surface through DNA hybridization For reagents and techniques used in this workflow refer to Bio Rad bulletin 6161 A 30 000 7 Liposome layer 4 Liposome layer 3 D a 20 000 Liposome layer 2 D a Cc Q Liposome layer 1 2 10 0004 A oc 04 I I I O 1 000 2 000 3 000 4 000 5 000 6 000 7 000 8 000 Time sec B 120 7 iii T 80 g ma Cc O 8 f 40 7 as AA atl ve O 4 200 0 200 400 600 800 1 000 Time sec Fig 2 10 ProteOn LCP sensor chip A sensorgram of the stable capture of four 1 palmitoyl 2 oleoyl sn glycero 3 phosphocholine POPC liposome layers Tagging complementary single stranded DNA molecules to the liposomes facilitates the multiple layer capture capability B sensorgrams of the interaction between FITC labeled 1 2 Distearoyl sn glycero 3 phosphocholine DSPC liposomes captured on the LCP sensor chip and an anti FITC antibody FITC labeled DSPC liposomes were captured to approximately 330 RU and the anti FITC antibody was injected
27. Propanolol 11 Verapamile 12 H Imipramine 13 E Desipramine 14 B Z 2 25 2 2 D D 15 3 14 O g s1 E E O T a Tl T T T oe 2951 1 2 3 4 5 6 T7 8 9 10 11 12 13 14 Drug number Fig 3 29 The results of the screening of 14 small molecule drugs against five liposomes using the 6 x 6 configuration in the ProteOn XPR36 system A sensorgrams obtained from the ligand channels of POPC and DSPC CHOL NH SO Weak or no responses were observed in the other ligand channels indicating no interactions between the small molecule drugs and the other three liposomes B normalized maximum signals of the interactions of the drugs and the liposomes Responses were normalized by dividing by the compound s MW Strong responses were observed when using POPC liposomes and ammonium sulfate gradient DSPC CHOL NH SO liposomes POPC m DSPC CHOL 67 33 m DSPC CHOL 55 45 m DSPC CHOL NH SO i DSPC CHOL PEG 50 45 5 m RU response units MW molecular weight Membrane Protein Analysis Using Lipoparticles Bronner V et al 2011 Highly efficient lipoparticle capture and SPR Willis S 2009 ProteOn XPR36 and lipoparticle technology A powerful binding kinetics of a membrane protein using the ProteOn XPR36 combination for screening antibody therapeutics against membrane protein interaction array system Bio Rad Bulletin 6161 teins Bio Rad ProteOn Webinar Series PICS PO eo Eee inal e
28. XPR36 system able to screen small molecules Which sensor chip is suggested for analyzing small molecules Yes The GLH chip has high ligand binding capacity in the amine coupling chips and the HTE chip has high capacity for capturing histidine tagged targets They are both designed for this type of analysis Are glycoproteins compatible with the ProteOn sensor chips Glycoproteins can be immobilized using amine coupling or captured on an NLC chip if the glycoprotein is biotinylated What are the differences between NTA and tris NTA surface chemistry in capturing histidine tagged proteins The nickel l activated nitrilotriacetate NTA surface chemistry is often used for capturing histidine tagged proteins in SPR experiments Tris NTA 3 x NTA surface chemistry is derived from NTA with improved capture stability and selectivity It results in minimal ligand drift and improves sensorgram baseline stability Compared to NTA sensor chips the tris NTA sensor chips allow easy surface regeneration chip reuse and capture of histidine tagged proteins directly from crude samples The Bio Rad ProteOn HTG and HTE sensor chips are tris NTA sensor chips What are the applications of ProteOn HTG and HTE sensor chips The HTG and HTE sensor chips feature a novel tris NTA surface for improved capture of histidine tagged proteins The functional group tris NTA is unique to the ProteOn HTG and HTE sensor chips and has a significantly higher b
29. a site specific tag ProteOn sensor chips such as the NLC HTG and HTE chips can be used The NLC chip is coated with NeutrAvidin for immobilization of biotinylated ligands and the HTG and HTE chips are coated with a unique and innovative tris NTA surface for immobilization of histidine tagged proteins sensor chips come packaged in a sealed pouch with an inert gas have a shelf life of two years if stored properly at 4 C and are guaranteed for six months from the date of receipt Sensor chips are continually monitored for quality and have excellent spot to spot reproducibility within the 6 x 6 interaction array Read more to learn about the different sensor chip types Figure 2 1 and which is best for your specific application 20 ProteOn Sensor Chips 2 3 Types of ProteOn Sensor Chips There are seven types of ProteOn sensor chips that can be used for a variety of different immobilization strategies and the creation of different capacity surfaces The GLC GLM and GLH sensor chips are designed for general amine coupling of ligands whereas the NLC sensor chips are designed for site specific attachment of biotinylated ligands and the HTG and HTE chips for histidine tagged proteins respectively Figure 2 1 shows an overview of the different sensor chip types and the specific applications in which they are used Chip Selector Compact capacity amine coupling for protein protein GLC l interactions GLM Medi
30. amino acid sequence facilitates the interaction between mutant and wild type AB peptides The observation is consistent with the previously reported molecular mechanism of AB aggregation Di Fede G et al 2012 Good gene bad gene New APP variant may be both Prog Neurobiol 99 281 292 Gobbi M 2012 Novel application of SPR to study amyloidogenic peptides and proteins Bio Rad ProteOn Webinar Series Stravalaci M et al 2010 Use of surface plasmon resonance to study the elongation kinetics and the binding properties of the highly amyloidogenic AB 1 42 peptide synthesized by depsi peptide technique Biosens Bioelectron 26 2772 2775 Taylor M et al 2010 Development of a proteolytically stable retro inverso peptide inhibitor of beta amyloid oligomerization as a potential novel treatment for Alzheimer s disease Biochemistry 49 3261 3272 These articles Di Fede et al 2012 Stravalaci et al 2010 and Taylor et al 2010 and the webinar Gobbi 2012 outline an SPR based approach to investigate the elongation of AB fibrils and an immunoassay to analyze the formation of AB oligomers The interaction between AB monomer AB oligomers and AB fibrils could be characterized with kinetic analysis using the ProteOn XPR36 system When AB fibrils were immobilized on the surface of sensor chips AB monomers and the mixture with inhibitors could be applied to investigate the elongation of AB fibrils and evaluate the inhibitors Us
31. and time consuming step Probing at one time six ligand immobilization conditions together with six analyte injection conditions the ProteOn XPR36 system allows for full optimization in a single run This ensures the optimal experimental conditions for the interaction between the ligand and the analyte The method of using a single run of 6 x 6 injections for a complete kinetic analysis is called the One shot Kinetics approach Kinetic Screening 6 to 1 In kinetic screening experiments each of the six ligand channels gives a full kinetic analysis in a single run This high throughput enables fast processing of a large number of samples while accurate kinetics is maintained The ProteOn XPR36 system provides the best balance between throughput and accuracy of kinetic screening Multiplex Screening 6 to 6 and Array Screening 36 to 1 In multiplex or array screening experiments the 6 x 6 interaction array of the ProteOn XPR36 system is fully utilized for high throughput which enables multiplex or 36 ligand screening 6 variations of a target 6 concentrations of the same analyte 6 different targets 6 concentrations of the same analyte 6 different targets 6 different analytes 60 60 600 00 36 different targets 1 analyte 14 ProteOn XPR36 Technology High Productivity Fast Protocol Optimization and High Throughput Sensor chip surface Kinetic Characterization ProteOn XPR36 system 1 1 h
32. avoid using a high activation level because it can lead to overactivation and subsequent multivalent linking of the ligand It is possible to eliminate the manual mixing of the activation reagents by using the Co inject function of the ProteOn XPR36 system Refer to the article Ligand Immobilization in Protein Interaction Studies An Unattended Amine Coupling Protocol with Automatic Co Injection Activation in the May 2012 online issue of BioRadiations Note 1x EDC sulfo NHS contains a 1 1 mixture of EDC and sulfo NHS components that are included with the ProteOn amine coupling kit Follow the instructions shipped with the kit to prepare the activation reagents The final concentrations of the activation reagents are 20 mM EDC and 5 mM sulfo NHS Ligand Immobilization It is recommended to prepare the ligand in a concentration range of 0 5 ug ml to 25 ug ml Typically using a slow flow rate of 30 ul min is suggested to reduce ligand use during ligand injection the contact time may vary from 1 min to over 10 min depending on the immobilization level needed The ligand coupling buffer is 1 pH unit lower than the ligand pl to facilitate charge attraction between the positive ligand and the negative chip surface Low ionic strength is also required to enhance charge attraction These conditions provide a starting point for further optimizing experimental conditions To achieve the desired immobilization level choose from th
33. buffer should match that of the sample buffer Correct for Bulk Effect 1 Apply proper referencing 1 Bubbles at the beginning of an injection are usually coupled with an injection delay in one or more channels Problems with bubbles during the injection step 2 Match the refractive index of the sample buffer and running buffer 2 Responses may vary in intensity among different 3 In experiments where analytes are dissolved in a ies channels injections and times cosolvent with a high refractive index such as DMSO the reference surface produces a larger bulk solvent response than the ligand surface because of the larger concentration of cosolvent near the reference surface This effect is due to the exclusion of cosolvent by the ligand near the ligand surface The resulting difference in bulk effect causes inaccurate reference subtraction To correct for the difference in bulk effect between 99 ProteOn XPR36 Experimental Design and Application Guide Techniques for Reducing Bubble Formation 1 Use prepierced vial caps and microplate sealing films to prevent vacuum formation inside the container during aspiration 2 Degas the sample and reagent solutions 3 Verify that the volumes of sample and reagent solutions are sufficient before injection 5 2 6 Regeneration Regeneration removes the analyte or ligand analyte complex on the chip surface to prepare the surface for the next experiment without damaging the
34. coating This versatile sensor chip is ideal for analyzing protein protein interactions Figure 2 2 A Analyte Incident light Detector Response RU Time min Fig 2 2 ProteOn GLC sensor chip A the thin alginate coating on the GLC sensor chip responsible for creating a compact capacity surface B sensorgrams of the interaction between the cytokine IL 2 and the anti IL 2 antibody using the GLC sensor chip The IL 2 antibody was immobilized to approximately 2 000 RU and IL 2 was injected in a twofold dilution series ranging from 80 2 5 nM The nearly planar surface of the GLC sensor chip allows for high quality kinetic analysis RU response units GLM Sensor Chip Medium Binding Capacity The GLM sensor chip is coated with a thicker alginate polymer that displays a higher amount of carboxylic acid groups and is thus ideal for creating medium capacity 12 KRU ligand surfaces via amine coupling It can be used for both protein protein interactions and protein small molecule interactions Figure 2 3 21 ProteOn XPR36 Experimental Design and Application Guide A Analyte Incident light Detector Response RU Time min Fig 2 3 ProteOn GLM sensor chip A the extended alginate coating on the GLM sensor chip responsible for creating a medium capacity surface B sensorgrams of a TEM1 B lactamase mutant interacting with the B lactamase inhibitor protein BLIP using the GLM s
35. constants can be derived These data are valuable to those studying biomolecular interactions in many applications from binding site interface analysis and concentration determination to thermodynamic analysis 4 3 2 Full Kinetic Profile To generate a full kinetic profile for the interaction of an analyte with a ligand and obtain the binding constants above one must measure the interactions at multiple analyte concentrations Typically multiple analyte concentrations are required for good model fitting The benefit to using the ProteOn XPR36 array system for kinetic analysis is that its 6 x 6 array lends itself perfectly to the simultaneous injection and analysis of up to six analyte concentrations at once One of these analyte concentrations can be sacrificed for use as a real time double reference 4 3 3 Determination of Analyte Concentrations As arough guide the range of concentrations needed for an analyte injection should span 10x greater than and 10x less than the expected K If you are starting with an unknown system and you have no prior knowledge of the K search the literature to discover if a similar interaction system has been previously studied to obtain guidance on where to start If this is not possible then consider what type of biomolecules you are working with For example if it is an antibody antigen interaction you would expect something within the nanomolar to subnanomolar K range for a tight interaction If t
36. details on achieving optimal ligand analyte interaction analysis Three useful tips for obtaining reliable interaction analysis results are listed below 1 Prepare the analyte samples as a concentration series typically a two or threefold dilution series centered around the expected K 2 If needed set up a double reference Replace one of the six analyte channels with running buffer for use as a real time double reference row reference Alternatively set up an injection of running buffer into all six analyte channels prior to the injection of analyte samples injection reference Double referencing is needed for baseline drift correction and is used mostly when the ligand is reversibly captured by a capture reagent such as an antibody NeutrAvidin NLC and LCP chips or a tris NTA complex HTG and HTE chips 3 Set the association time of the interaction to be long enough to observe curvature in the association phase Similarly allow the dissociation time of the interaction to be long enough to observe a signal drop in the dissociation phase The concentration range of analyte should span 10x above and 10x below the expected K A buffer blank can also be injected as a real time double row reference Note In protein small molecule interaction analysis the small molecule analyte is sometimes prepared with a high refractive index cosolvent such as DMSO In such a case excluded volume correction is typically used
37. eee Dee Aaro e Tho Ctrl key to select multiple graphs or press Ctrl A to select all graphs 3 Right click any selected graph and choose Copy Data A status bar is shown When the copy process is completed paste the graph into a spreadsheet The first column lists time values and the other columns list the sensorgram response values and the corresponding curve fitting data points 3 Inthe menu bar select File gt Export Document and choose the report format in the submenu Click OK in the dialog box select the target folder in which to store the report and click Save to save the experiment report 86 Experimental Design 87 Tips and Techniques CHAPTER 5 The tips and techniques for using ProteOn sensor chips including experimental conditions and troubleshooting guides are essential for successful surface plasmon resonance SPR experiments The tips and techniques are organized by the surface chemistries of ProteOn sensor Chips ProteOn XPR36 Experimental Design and Application Guide 5 1 Tips for Using ProteOn sensor Chips All ProteOn sensor chips are designed for use with the ProteOn XPR36 protein interaction array system Each sensor chip is a gold coated glass prism containing surface chemistry used for the immobilization of biomolecules ligands to the chip surface This enables interactions with other biomolecules analytes to create a measurable SPR response used in kinetic analys
38. he standard analyte response that gives the best kinetic analysis is between 100 200 RU RU response units 63 ProteOn XPR36 Experimental Design and Application Guide Choosing a Sensor Chip for Amine Coupling Which sensor chip is used to study the interaction will depend on the level of immobilization of ligand that is required and on the specific application A complete guide to the different ProteOn sensor chips is given in Chapter 2 In short for protein protein interactions GLC and GLM chips are sufficient For protein small molecule interactions GLM and GLH chips are the best choice 4 2 4 Deactivation The injection of 1 M ethanolamine follows the ligand immobilization step and deactivates any unreacted sulfo NHS ester groups Default injection parameters are 30 ul min for 5 min 4 2 5 Stabilization This step is generally performed to ensure that any noncovalently attached proteins that may still be electrostatically held at the sensor surface are removed prior to the analyte injection and interaction analysis Stabilization buffer is injected across the surface The type of buffer ranges from running buffer to harsher solutions like 50 mM NaCl and 50 mM NaOH Care should be taken not to use a stabilization buffer that is so harsh that the immobilized ligand is denatured This will reduce its activity affect the interaction analysis and reduce analyte response Use short injections of 80 60 sec with high flow rates
39. layer Analyte Surface layer Gold substrate Gold substrate Fig 5 5 Non electrostatic NSB on the chip surface Table 5 6 Molecules with a propensity for NSB Techniques for Reducing or Eliminating NSB on All Sensor Chips Molecules in Samples Type of NSB Suggested changes in experiment design to reduce Positively charged proteins with a pl gt pH of the running buffer Relatively small molecules containing 1 Optimize the appropriate running buffer and sample thiol groups or related forms of sulfur such buffer for the application as cysteine containing peptides l known to show high affinity to gold 2 Purify the analyte or sample when possible Electrostatic NSB NSB Non electrostatic NSB Molecules that naturally bind 3 Try different immobilization surface chemistries polysaccharides such as lectins may exhibit NSB to the alginate containing Nonm eeairostatig NE for example use a biotinylated ligand binding layer 4 Swap the ligand and the analyte if only the analyte Biological solutions such as serum crude Electrostatic NSB and shows NSB lysates or supernatants Non electrostatic NSB 5 Retest your binding assay with a fresh chip preferably from a different lot to eliminate chip specific defects 98 Tips and Techniques Table 5 7 Techniques for reducing or eliminating NSB on all sensor chips Non electrostatic Methods Details NSB Electrostatic NSB Increase the salt con
40. of cosolvent near the chip surface caused 2 i 100 DMSO by the exclusion by the ligand of cosolvent from the chip Final Volume 10 10 2 000 2 0 surface of the interaction spot This is known as the excluded volume EV effect Figure 4 24 o Bs D Ske solution Concentration v d Analyte uM 20 10 5 2 5 1 25 0 625 DMSO 5 5 5 5 5 5 Wrong signal Binding Fig 4 25 Analyte preparation Dilute the stock analyte solution with the highest concentration of analyte using the freshly prepared DMSO running buffer The analyte concentration will be reduced but the Fig 4 24 Explanation of the EV effect when using cosolvents with DMSO concentration will stay the same for example DMSO 5 PBS high refractive index such as DMSO Normally the bulk effect will be cancelled out after reference subtraction However the bulk effectisnot 5 Flush the instrument twice with the interaction equal on both the active and reference spots due to DMSO exclusion by analysis buffer containing the cosolvent at the the ligand from the surface on the active channel i vs oreferred concentration in buffer position A Small differences in the concentration of DMSO in the 6 Prepare six different concentrations of DMSO in analyte and running buffers also lead to large changes fresh running buffer Figure 4 26 in response This bulk effect is 100 RU for every 0 1 difference in DMSO concentration esses Performing an EV corr
41. reagent hT YF A ii ii ii I I i Surface ll l Surface 2 ee 2 B Protected immobilization method Protected ligand Surface cc Surface Analyte Ligand p Ligand Surface M M M E Fig 5 2 The capture surface method A and protected immobilization method B for higher ligand activity Surface 95 ProteOn XPR36 Experimental Design and Application Guide Ligand Capture with the NLC Chip Tips for capturing ligands with NLC chips 1 It is important to assess the result of biotinylation after ligand preparation If the biotinylation step was not successful ligand capture will not be observed on NLC chips 2 If excess biotin is not removed properly from the ligand sample it will occupy the available binding sites on the chip surface and result in low binding levels typically of a few tens of RU 3 If the ligand is biotinylated and captured on the chip surface but does not show a binding response with the analyte over biotinylation may have occurred To avoid this prepare the ligand in a stoichiometry of 1 1 one biotin molecule per ligand molecule This also prevents cross linking of the ligand Alternatively carry out the biotinylation reaction in a low pH buffer 50 mM acetate at pH 5 5 to favor the selective biotinylation of aloha amino groups which leaves the lysine residues unblocked 5 2 4 Stabilization Stabilization is the step between ligand immobilization and analyte injection Injecting ru
42. robust data The ProteOn XPR36 system is a multiplexed SPR instrument that utilizes novel fluidics to monitor the interaction of up to six ligands and six analytes This allows for the simultaneous study of up to 36 interactions on the surface of the sensor chip greatly increasing experimental throughput and reducing assay develooment time In the simplest SPR experiment using the ProteOn XPR36 system a ligand is covalently immobilized to the surface of the sensor chip and interacts with an analyte present in the running buffer that flows over the surface of the sensor chip This is known as direct immobilization In another commonly used method a biomolecule is used to capture the ligand prior to analyte interaction In this case the ligand is not covalently immobilized on the chip surface but is captured through biomolecular interactions The advantages of ligand capture are as follows Creates a homogenous ligand surface well defined orientation Purifies ligand on the chip surface Allows regeneration of the ligand surface Both methods have different advantages depending on the type of interaction analysis that is being carried out The major steps of each method are discussed in this guide Part 1 Direct Immobilization of Ligand Methods of direct immobilization include amine coupling thiol coupling and aldehyde coupling While all these methods are applicable on the general use GLC GLM and GLH chips the a
43. the ProteOn protein small molecule kit In this experiment six identical ligand channels were prepared so that the global fitting of all 36 sensorgrams and the grouped fitting of a set of six Sensorgrams in each ligand channel are comparable Table 4 3 Results of an experiment using the ProteOn protein small molecule kit Parameter k 1 Ms k 1 s Kp M R RU Chi RU Scope Global Global Global Global All Six 10 363x107 2410 80 52 6 0 Scope Grouped Grouped Grouped Grouped All L1 158x10 370X107 206X10 84 9 5 3 L2 1 60 x 104 3 75x10 235 410 18 7 5 0 L3 iSo Sox Gan 20x 0e 79 3 5 1 L4 1 54 x 104 3 54x10 2 30 x 108 78 6 5 8 LS 142x10 860x102 251 x 10 80 4 5 8 L6 1 36 x 104 3 48 x10 2 56x 10 81 3 8 1 Global Parameters are identical for all sensorgrams Grouped Parameters are identical for a certain ligand channel 4 5 2 Equilibrium Analysis The equilibrium constant Kp can be calculated directly from a sensorgram using Equation 9 Ps A R _ TK Al Equation 9 Equation 9 describes the response at the steady state or equilibrium phase of the interaction as shown in Figure 4 16 In this phase the rate of association equals the rate of dissociation To determine the Kp the response at equilibrium R is measured over a given range of analyte concentrations and the values are plotted as shown in Figure 4 17 Req is proportional to the analyte concentration at the low concentration
44. the analysis of multiple experimental conditions in a single experiment Figure 3 34 54 Applications Human IgG1Aa IL 6 cytokine IL 6 antibody Protein A Protein A G 200 5 4 000 as 2 700 4 ag Kaponiera gann D 2 2 000 U Fa ug co gt S 1 100 5 S oc D D S 100 J am T T T T am 0 T T T T 2 Y Ei tlie acne iat Astotais 80 360 640 920 80 360 640 920 9 d y Time sec Time sec ikiia Protein L Protein G ry Teak ihe aoe oio 46 4 000 Oo a T E 0 2 4 6 8 10 12 167 2 2 000 Time min 5 S E g o IL 18 cytokine IL 18 antibody 80 360 640 920 80 360 640 920 200 Time sec Time sec gE Worn P A emcee O O Fig 3 34 Screening of human IgG1A to the immunoglobulin gt Fa ie ecient binding proteins RU response units iF inte ee e i e n h aa alae 1004 JF 4 ee S Fa 3 ay cana semmen mere TT OT Bronner V et al 2006 Rapid and detailed analysis of multiple antigen 50 4 IFA _ antibody pairs using the ProteOn XPR36 protein interaction array Pe yr al RAEDT PEA system Bio Rad Bulletin 5360 o aa f f f This technical note Bronner et al 2006 describes 0 2 4 6 8 10 12 the rapid and detailed characterization of four Ag Ab Hiona mia interactions by the ProteOn XPR36 system to fully exploit Negative control TEM1 the 6 x 6 interaction array Four different antibody targets 50 and a neg
45. turn yellow table and select the correct sample If needed adjust D Launch ProteOn Manager software the step setting such as Flow Rate Contact Time Wait until all five instrument LEDs and Communication state in the software turn green E Select Protocol Check and review the protocol steps F If the instrument is started after long term shutdown follow the pop up instruction in the software to flush F the fluidic system If a printed copy of the protocol or the sample layout is needed for sample preparation select Protocol 2 Initialize the sensor chip Report or Sample Report Note If the sensor chip to be used is already in the instrument start 2 Run a protocol from step D if it is not initialized Press Resume if the Instrument State A Prepare and load the samples in the sample is in Standby container The sample container must be either a A In the Navigator panel go to the Instrument tab rack or microplates and consistent with the sample and select Instrument Control container information in the Configuration screen Place the sample container in the instrument correctly positioned with sample vial well A1 at bottom left corner B If there is a sensor chip in the instrument press Eject to eject it C Insert the sensor chip to be used Wait until the Initialization Status box shows Chip Not Initialized B In the Navigator panel go to the Run tab and select Ps ees the protocol in the Se
46. via incubation in an acidic buffer Activation of carboxyl groups for ligand immobilization is done using carbodiimide chemistry with the reagents 1 ethyl 3 6 dimethylaminopropy carbodiimide EDC and N hydroxysulfosuccinimide Sulfo NHS Once activated the resulting sulfo NHS esters are highly amine reactive and react with free amines exposed on the ligand to immobilize it to the sensor chip Any unreacted carboxyl groups that remain activated during the immobilization step are deactivated with ethanolamine to prevent immobilization of analyte protein during the subsequent interaction step The chemical structure of the binding layers forms easily activated carboxyl groups rendering especially high binding capacity and ligand activity It results in more active ligand on the surface thus higher analyte signals and higher assay sensitivity Different amounts of ligand may be amine coupled to the GLC GLM and GLH sensor chips by controlling the amount of ligand in solution during the immobilization by tuning the activation level or by adjusting the length of time of the immobilization step The amount of ligand bound is monitored by following the binding response displayed on the sensorgram in real time However specific applications may require a surface with a very high or low ligand surface capacity In such cases GLH and GLC chips that have a very high or low density alginate coating may be used For immobilizing targets through
47. washed off in dissociation causing exponential drift Fig 1 7 Optimal experimental conditions are obtained in a single run Fig 1 8 Novel ProteOn XPR36 system references The ProteOn XPR36 system provides A an interspot blank surface reference to save interaction spots and provide immediate proximate referencing and B a real time injection reference to correct the exponential baseline drift when using ligand capture surface chemistry 16 ProteOn XPR36 Technology 4 Data Analysis Software Advantages ProteOn Manager software is a comprehensive user friendly tool for the analysis of biomolecular interactions Features include Ease of use Integration of data acquisition data processing and data analysis Powerful graphic user interface Intuitive protocol writing interface Fast and accurate data processing Accurate fitting with 8 models Rapid data analysis Concise analysis reports Export functions for further data processing in Excel or other software Ge Edit Yew Process ndyo Jools Help DA PAAM D At 1 O S gate Dbperet OCMC Marg Tert U01 n EEEE mee 6 2 IEN OEO u g i oo w oa ee ee a Tower 5 Towe s esmase i i i aH 6 20 De ee me a mea 5 2 Ow i wh te we oe T in Towe ed Fig 1 9 ProteOn Manager software data analysis window 1 5 ProteOn Webinar Library The ProteOn webinars feature presentations from thought leaders in the field of label free biomolecul
48. widely used tags in protein purification Recombinant proteins containing the histidine tag are easily captured by an NTA surface Bio Rad offers the HTG and HTE chips for capturing histidine tagged proteins for interaction analysis The chips employ a novel tris NTA surface chemistry which provides higher stability and selectivity in capturing histidine tagged proteins compared to the traditional NTA surface chemistry and allows for surface regeneration This surface intercalates at three points with a histidine tagged protein rather than a single point for the NTA surface Figure 3 24 The HTG chip is ideal for the analysis of protein protein and protein peptide interactions and HTE chip for protein small molecule interactions Figure 3 25 The technical notes and the poster describe both protein protein and protein small molecule interactions performed using the HTG and HTE chips A Comparison of NTA and tris NTA binding to histidine tagged proteins NTA p Traditional surface Tris NTA Novel surface B Protein Histidine 6 Ni NTA Percent of protein that remained bound 5 min after end of injection Tris NTA NTA Protein A 100 97 Protein A G 96 88 Ubiquitin 92 45 Fig 3 24 Description of tris NTA technology A 300 or 5 200 Cc O S A 100 ow ree YEO EN At 8 a 0 200 100 O 100 200 300 400 500 600 700 800 Time sec oO A
49. with high binding capacity ProteOn LCP sensor chip for capturing lipid assemblies such as liposomes for use with LCP capturing reagent kit On a single SPR sensor chip can I use one spot channel at a time for ligand immobilization and reserve the blank spots channels for a future experiment Yes it is possible to immobilize ligands in individual spots channels with any of the amine coupling histidine tag capture and lipid assembly capture sensor chips This may not apply to streptavidin or NeutrAvidin sensor chips if an additive is used to stabilize these proteins on the surface 104 Frequently Asked Questions Can I reuse a spot channel after a ligand is immobilized to the surface of the sensor chip It is possible to reuse a spot channel after a ligand has been immobilized to the surface of the sensor chip if the chip surface is regenerated and preserved properly to keep the ligand activity How many times is it possible to regenerate a sensor chip surface The extent of regeneration depends on the immobilized protein ligand In the case of small molecule screening running buffer is often the regeneration solution Therefore the protein ligand remains quite stable and active for multiple rounds of analyte injections However when an acid base or detergent is required for regeneration the ligand may lose its activity and a positive control is recommended to monitor the ligand activity Is the ProteOn
50. 0 X axis title anne Y axis title O Specify fit color aeey Copy Graph Size Reduced for printing and presentation O Full size Fig 4 21 Examples of an isoaffinity graph top and a screening graph bottom 4 5 6 Sensorg ram Appearance Fig 4 22 The sensorgram smoothing and sensorgram appearance oo setting functions in ProteOn Manager software To customize the sensorgram visualization to meet user preferences ProteOn Manager software offers Note Please refer to section 4 8 for the different options of exporting two appearance functions sensorgram smoothing and SPR results to external software platforms from ProteOn Manager l software sensorgram appearance setting Sensorgram smoothing is available in the View menu and may be removed by deselecting it This function smoothes the baseline noise to better display the curvature of sensorgrams Note that the sensorgram smoothing is only a display related feature It does not affect sensorgram fitting or SPR results because the data analysis is always based on the raw data The sensorgram appearance setting is available in the Tools menu and allows the user to choose sensorgram color and line thickness The appearance change will be applied to the sensorgram series a particular sensorgram in all sensorgram sets rather than a single sensorgram To change the color of a single sensorgram open the Interaction screen in the Data tab and right click an interacti
51. 0 mM HCI Horizontal 30 30 4 300 mM EDTA Horizontal 30 30 5 0 5 SDS Vertical 30 30 6 50 mM NaOH Vertical 30 30 7 100 mM HCI Vertical 30 30 8 300 mM EDTA Vertical 30 30 Note 1 In conditioning it is recommended to use the same buffer used for running experiments When working with buffers containing metal ions that form hydroxide precipitates however eliminate the NaOH injections 2 Trehalose is used as a protective layer for the dry NeutrAvidin on the NLC chip surface It will be completely removed by continuous buffer flow over the chip surface However conditioning is highly recommended to ensure complete removal of the protective layer 3 For the LCP chip used with the ProteOn liposome capturing kit perform conditioning after biotin ssDNA capture and before liposome capture For the GLC chip used with the ProteOn GLC lipid kit perform conditioning after surface modification and before liposome capture Refer to Section 5 2 2 Ligand Immobilization for more details 91 ProteOn XPR36 Experimental Design and Application Guide 5 2 2 Ligand Immobilization Ligand immobilization refers to the attachment of a ligand to the chip surface either by irreversible covalent bonding or by reversible capture using a capture reagent Refer to Chapter 4 section 4 2 for details on achieving optimal ligand immobilization Desired Ligand Immobilization Level The immobilization level R or amount of ligand Immobilized on the chip surfa
52. 00 O 200 400 600 800 200 O 200 400 600 800 Time sec Time sec B CD81 Antibody Capture E MemLAYER Capture Double Layer 80 250 r Aten Y itm aA 200 60 a if Tae Orn TOR A I LANEAN IT eo Ra A ells hs yey bor Ms Wi 150 40 lj i AAN Oe me 2 I Kha 2 100 z yf WAN WaltA AR S Q 204 AN OY 7 g E V MTA Aw iam 50 4 O Di O z 20 50 200 0 200 400 600 800 200 0 200 400 600 800 Time sec Time sec C Undecylamine Capture 300 250 200 150 no C 9 4100 op O oO 50 O 50 200 O 200 400 600 800 Time sec Fig 3 30 Kinetic analysis for comparing the different lipoparticle immobilization approaches CXCR4 lipoparticles were immobilized on GLM using the four described methods followed by One shot Kinetics injection of CXCR4 antibody A through E show the sensorgrams for the five analyte concentrations with the overlaid 1 1 model fit 30 nM m 15 nM m 7 5 nM m 3 75 nM m 1 875 nM m RU response units 51 ProteOn XPR36 Experimental Design and Application Guide Identification of Membrane Protein Interactions Through Domain Screening Jiang L et al 2010 Identification of leucocyte surface protein interactions by high throughput screening with multivalent reagents Immunology 129 55 61 This article Jiang et al 2010 describes a method to screen the interactions between membrane protein extracellular domains It uses the 36 ligand ar
53. 10 100 20 60 140 220 300 100 20 60 140 220 300 100 20 60 140 220 300 Time sec Time sec Time sec 1e 1 1 Kp nM k M s ky S Kinase B 8 1 75 x 108 1 4 x Ore Kinase C 34 1 60 x 10 5 5 x 107 Inhibitor B ATPase Kinase A Kinase B 25 T T 18 T a g 11 A N N N C Cc Cc O o 4 O Q Q Q n 2 _3 n T x x 10 100 20 60 140 220 300 100 20 60 140 220 300 100 20 60 140 220 300 Time sec Time sec Time sec Kinase C Kinase D Kinase E z A a e e O O Q Q 2 a T 100 20 60 140 220 300 100 20 60 140 220 300 100 20 60 140 220 300 Time sec Time sec Time sec 1a 1 1 Kp nM k M s ka S Kinase C 8 4 3 9 x 10 3 2 x 1078 Fig 3 11 Screening of two inhibitors binding kinetics to the six different ligands Inhibitors A and B concentrations are 1 000 333 111 37 and 12 uM RU response units 37 ProteOn XPR36 Experimental Design and Application Guide Tabul M et al 2010 Rapid high throughput screening of protein kinase Compounds from two small molecule kinase inhibitor inhibitors using the ProteOn XPR36 protein interaction array system libraries were screened against each kinase target to Bio Rad Bulletin 5965 eae identify binding hits Figures 3 12 and 3 13 Detailed kinetic analysis of the positive binding compounds was conducted to determine the binding constants Screening of 110 compounds took only 5 hr and all the work was completed o
54. 2014 SPR as a new technology in clinical research Bio Rad ProteOn Webinar 2014 Series A Infliximab N 000 0000 TNF Surface Ligand TNF Analyte Infliximab Density 2000 2500 RU Spiked serum samples pH 5 0 Patient samples Concentation 5 ug ml B 800 600 7 5 a T Initial rate oO e 5 400 NN Q op 0 or 200 7 O Vo aie 40 O 40 80 120 160 200 Time sec Normalized Rate l 0 1 1 10 Concentration of Infliximab ug ml 100 Fig 3 37 Quantitation of infliximab in serum samples A illustration of the SPR surface chemistry B quantitation based on the initial rate of the ligand analyte interaction E 20 ug ml m 10 ug ml m 5 ug ml m 1 ug ml 0 6 ug ml m 0 3 ug ml C the initial rate obtained from standard analyte samples is plotted against analyte concentrations to form a standard curve This webinar highlighted how the ProteOn XPR386 system was used to quantitatively measure the serum levels of the TNFa inhibitor drug infliximab Infliximab is a chimeric monoclonal antibody that targets TNFa and is used to treat a variety of chronic autoimmune disorders Monitoring serum infliximab concentrations is important in guiding management especially when a patient is not responding well to treatment Infliximab concentrations can determine if loss of efficacy is due to an inadequate dose or to the development of anti infli
55. 9 disulfonamide Benzenesulfonamide 157 50 1 17 x 10 0 12 1 0 x 1078 114 7 fluoro 2 1 3 benzoxadiazole 217 2 4 64 x 10 0 01 2 8 x 10 8 82 4 sulfonamide Acetazolamide 222 2 9 28 x 10 0 02 2 6 x 108 99 Methylsulfonamide 95 2 500 3 2 x 1074 22 36 Applications Screening of Kinase Inhibitors small molecule kinase inhibitors against five known Miura Tet al 2010 High throughput profiling of kinase inhibitors important kinase targets The biotinylated kinases and selectivity using the ProteOn XPR36 protein interaction array system Bio Rad Bulletin 5980 one Al Pase were immobilized and a concentration series of the inhibitors were injected Figure 3 11 The assay was evaluated using Z factor analysis and it was shown that an R_ of 17 RU was sufficient to achieve consistent results This technical note Miura et al 2010 describes the use of the One shot Kinetics approach of the ProteOn XPR386 system to generate K values during the screening of Inhibitor A ATPase Kinase A Kinase B oO O ON O 2 38 z 38 T g 26 4 g 26 3 o 14 o 14 O 3 3 3 10 i i i l l 10 T T 10 Ta T T T 100 20 60 140 220 300 100 20 60 140 220 300 100 20 60 140 220 300 Time sec Time sec Time sec Kinase C Kinase D Kinase E 50 50 T Z 38 z 38 g 26 f 26 S S 14 S 14 3 F M 3 2 2 10
56. A good recommendation for ligand stock buffer is 0 5 1 mg ml Avoid or minimize any other amine containing compounds or any strong nucleophilic groups such as azide or Tris buffer as these amines will compete with the ligand amines Guidelines for Immobilization Levels What level of ligand immobilization to use depends on the type of interaction under study However less is more is a good guide and this is generally followed for kinetic binding measurements With a high density surface mass transport issues and crowding effects may result in altered kinetics See Table 4 1 An easy way to help determine which ligand level to use is to calculate the theoretical R nax of the interaction to be studied Using Rmax to Determine Ligand Immobilization Levels The theoretical Rna is the maximum analyte response assuming all of the ligand is active ligand is 100 pure and all binding sites are available When using amine coupling assume that not all ligand binding sites will be available after immobilization since this is a random coupling of the ligand to the sensor chip and therefore the ligand is not present in a homogenous orientation at the sensor chip surface see Figure 4 2 MW Rie A max MW xR xn Raye Maximum theoretical response of the analyte for a given ligand level R amount of ligand immobilized MW molecular weight n stoichiometry of the reaction Fig 4 2 Determining theoretical Rmax
57. OSHT 100600 6195 05 9 9605 140 85 LSE 10 625 Lia 17a 2 POSHT S 006 09 6196 05 9 5605 140 85 148 10 2 87 tias wy IL PESHT 2 50609 6196 05 9 50605 140 85 1 546 10 3 08 RA 172 IL PESHT 4 00E 08 5 246405 904E05 178 60 1 736 10 16 23 L2a2 vu IL POSHT 200600 S 28E 0S 9005 178 60 173 10 43 08 Laas 17 i IL2 POSHT 1 006 00 5 246405 901605 178 60 1 736 10 8 27 LAA wy IL PESHT 500609 S5SME 05 J0E05 178 60 1 736 10 3 25 LAS 172 IL PESHT 2 506 09 5 246405 904E05 178 60 1 736 10 5 31 Gal wu IL POSHT 4006 08 GA2E 0S 9 79605 HA LS2E 10 3 00 LA 17 ia IL2 PESHT 2 006 00 426 05 9 75605 ba DY L S2E 10 2 73 LRA va ILI PESHT 1 006 08 6426 05 9 7605 at 1928 10 3 42 had 172 IL PESHT S 00E 09 8 G6 A2E 0S 9 786 05 281 152 10 3 37 Laas vw IL POSHT 20609 GAES 9 70605 HA LS2E 10 2 4 LSAL 172 IL2 POSHT 4008 08 GOOS 2274 43 05 3428 10 JH LSAD 1732 12 PESHT 2 006 08 8 643E 0S 2 2784 43 85 3AQE 10 2 77 t53 na IL PESHT 1 006 08 6636 05 227E 43 85 3428 10 9 78 saa vue IL POSHT S00E09 GGS 206M 43 85 3 42E 10 Ja LSAS 17 i2 iL2 POSHT 2508 09 G6I 0S 2270 0 33 085 3 428 10 8 97 L AL wa ILI PRSHT 4005 08 6726 05 1 PE 14 95 2566 10 3 81 L a 1732 12 PBSHT 2006 08 6 72E0S LPEN 14 95 2468 10 an as 1732 IL POGHT 100600 672405 173 4 14 95 2466E 10 2 22 LOA4 172 12 POSHT 5008 09 6 720 0S 1730H 14 95 246 10 2 08 LOLS vw 12 PRSHT 2 506 09 6 725405 17E 14 95 2666 10 3 43 Fig 4 20 The grouping and ungrouping of the data table
58. Protein Interaction Analysis Nig dod ProteOn XPR36 Experimental Design and Application Guide ProteOn XPR36 Experimental Design and Application Guide Introduction 5 4 2 7 Ligand Capture by Biotin Label or Histidine Tag The NLC HTG and HTE Sensor Chip 64 Chapter 1 ProteOn XPR36 Technology 9 4 2 8 Summary 65 1 1 ProteOn XPR36 Technology Overview 10 4 3 Guide to Analyte Interaction 1 2 What Kind of Information is Obtained AE n XP36 System i m PJ with the Prater XPR36 System 11 Ve K erotic 66 1 3 How are Kinetic Parameters Obtained 12 4 3 3 Determination of Analyte Concentrations 66 1 4 Advantages of the 6 x 6 Interaction Array 13 4 3 4 Analyte Preparation 66 4 3 5 Analyte Injection Parameters 66 1 5 ProteOn Webinar Library 17 4 3 6 Analysis of Binding Results 67 Chapter 2 ProteOn Sensor Chips 19 4 4 Guide to eet oe s8 on the ProteOn XPR36 System on Naraw ad 4 4 1 Interaction Sensorgram Terms 68 2 2 ProteOn Sensor Chip Surface Chemistry 20 4 4 2 Sensorgram Display 68 2 3 Types of ProteOn Sensor Chips 21 4 4 3 Sensorgram Processing 69 2 3 1 Amine Coupling ProteOn Sensor Chips 4 4 4 Sensorgram Referencing 70 GLC GLM and GLH 21 4 4 5 Quality Standards for Processed Sensorgrams 72 2 3 2 ProteOn Sensor Chips for Site Specific Attachment 4 5 Guide to SPR Data Analysis NLC HTG and HTE 23 on the ProteOn XPR36 System 73 2 3 3 ProteOn Sensor Chips for Capturing Lipid 4 5 1 Kinetic Analysis 73 Assemb
59. acting with one 2 uM TEM1 mutant protein ligand Black lines represent the global fit of the sensorgrams to a 1 1 kinetic interaction model See Table 3 2 for the kinetic constants derived from these data RU response units Table 3 2 Kinetic and equilibrium constants for the interactions between BLIP and TEM1 mutants TEM1 Mutant k M sec7 k sec Kp nM R243A S235A 1S x 108 5 09 x 1074 33 8 R243A S130A 1 27 x10 1 24 x 1073 97 6 S130A S235A 3 10 x 104 9 33 x 1074 30 1 K234A 2 01 x 104 8 50 x 1074 42 3 E104A 1 70 x 10 7 40 x 107 43 5 41 ProteOn XPR36 Experimental Design and Application Guide Characterization of the B Amyloid Peptide Assemblies in Alzeheimer s Disease Research Di Fede G et al 2009 A recessive mutation in the APP gene with dominant negative effect on amyloidogenesis Science 323 1473 1477 This article Di Fede et al 2009 reports the characterization of an amyloid precursor protein mutation A678V that causes disease only in the homozygous state whereas heterozygous carriers are unaffected The ProteOn XPR36 system was used to determine the binding of wild type and mutated AB 1 40 or AB 1 6 to AB 1 40 fibrils The results showed no difference between AB 1 40 and AB 1 40 nu binding to AB 1 40 fibrils However the amino terminal fragment AB 1 6 Showed greater ability to bind to AB 1 40 fibrils than did AB 1 6 indicating that the A to V substitution in the
60. ad Bulletin 5358 Reichmann D et al 2005 The modular architecture of protein protein binding interfaces Proc Natl Acad Sci USA 102 57 62 This technical note Bronner et al 2005 and article Reichmann et al 2005 employ the ProteOn XPR386 system to uncover important residues for the interaction of two proteins TEM1 beta lactamase TEM1 and its inhibitor beta lactamase inhibitor protein BLIP In this study double mutant cycle DMC analysis was used along with the innovative One shot Kinetics approach in the ProteOn XPR36 system The DMC analysis is an excellent tool to investigate the structure mechanism and dynamics of protein protein interactions Multiple mutants of TEM1 and BLIP were analyzed against each other to determine the contribution of residues toward the stability of the TEM1 BLIP interaction Bronner V et al 2005 Analysis of multiple protein protein interactions using the ProteOn XPR36 protein interaction array system Bio Rad Bulletin 5368 This technical note Bronner et al 2005 employs the ProteOn XPR36 system to uncover important residues for the interaction of two proteins TEM1 beta lactamase TEM1 and its inhibitor beta lactamase inhibitor protein BLIP Figure 3 16 In this study the interaction analysis of TEM1 mutants and BLIP was used to construct a picture of the TEM1 BLIP binding interface The innovative One shot Kinetics approach in the ProteOn XPR386 system was employed to achieve kineti
61. aminants in the analyte sample If a ligand sample contains albumin inject a pulse of 1 M salt solution to remove the albumin bound to the chip surface before e the analyte injection Remove albumin from the ligand sample Note Refer to Bio Rad bulletin 6302 for specific troubleshooting tips for HTX chips Bulk Effect interaction and reference surfaces excluded volume The bulk effect refers to a spurious SPR response that correction is applied refer to Chapter 4 section 4 6 for is caused by changes in the refractive index of the more details solution near the sensing surface rather than the binding Bubble Formation of biomolecules to the surface Such refractive index Separation air bubbles are intentionally created between changes typically occur during the sequential injection the sample and running buffer to prevent mixing during of two solutions with different compositions such as sample aspiration No bubbles should be injected into different salt detergent or biomolecule concentrations the ligand or analyte channels Spikes in the sensorgram A small bulk effect can be completely removed by usually indicate the injection of bubbles Small spikes applying proper referencing but a large bulk effect can be completely removed in sensorgram processing may cause inaccuracy in data processing and analysis but large spikes may cause inaccuracy in experimental To minimize the bulk effect the refractive index of the results running
62. ar interaction analysis who use surface plasmon resonance SPR technology Visit www bio rad com info proteon to view Bio Rad s extensive library of ProteOn webinars and to sign up for future webinars References Abdiche YN et al 2011 Expanding the ProteOn XPR36 biosensor into a 36 ligand array expedites protein interaction analysis Anal Biochem 411 189 151 Daghestani HN et al 2010 Theory and applications of surface plasmon resonance resonant mirror resonant waveguide grating and dual polarization interferometry biosensors Sensors 10 9630 9646 17 ProteOn Sensor Chips CHAPTER 2 Used with the ProteOn XPR36 system ProteOn sensor chips are built with an alginate polymer matrix or a self assembled monolayer of organic molecules bound to a thin gold film on a sensor prism The sensor chips can be functionalized with several different reactive groups to achieve a variety of immobilization surface chemistries The surface chemistry of the ProteOn sensor chips allows the ProteOn XPR36 system to detect tight binding interactions down to picomolar concentrations of analytes or analytes as small as 95 daltons Da The combination of ProteOn sensor chips with the unique 6 x 6 interaction array allows the interaction analysis of up to 36 separate ligand analyte pairs on a single chip thereby increasing the throughput of a single experiment ProteOn XPR36 Experimental Design and Application Guide 2 1 Overvi
63. ar phase of the response is measured Concentrations are too high most of them are saturating and excess sample is used Fig 4 3 Analyte concentrations and injection length Extracting reliable binding kinetic constants requires 1 the use of several analyte concentrations that bracket the K value and 2 injection length long enough to see a curvature of the binding response 66 Experimental Design Initial analyte injection times can be guided by the strength of the interaction you are examining If you are working with a small molecule that has fast binding and dissociation from the ligand surface then you can keep the times short between 1 2 min association and 1 2 min dissociation See the table below for guidance on analyte injection times Table 4 2 Analyte injection time Association Dissociation Type of interaction min min Fast binding fast dissociation 1 2 1 10 Slow binding slow dissociation 5 10 60 Fast binding slow dissociation 1 2 10 60 4 3 6 Analysis of Binding Results Once you have optimized the analyte injection parameters and concentrations to generate good quality reproducible data you are ready to perform data processing and analysis This analysis allows you to determine the type of interaction you are dealing with and to obtain binding kinetics concentration or thermodynamic parameters Please refer to sections 4 4 and 4 5 67 ProteOn XPR36 E
64. ass transport control using a low flow rate and or a high capacity sensor chip bearing an analyte specific ligand Under such mass transport limited conditions the association rate of binding or the initial binding rate is proportional to the concentration of analyte in solution The concentration of analyte in a crude sample can be calculated by comparing the initial binding rate to a standard curve of initial binding rates for known concentrations Apply up to 6 unique target molecules such as mutant or wild type proteins 1 3 How are Kinetic Parameters Obtained By fitting sensorgram data from a ProteOn XPR36 system experiment to a suitable binding model kinetic parameters such as the association rate constant k dissociation rate constant k and the equilibrium or affinity constant K can be extracted Kinetic data are crucial for characterizing an interaction as they allow for a thorough understanding of the nuances of binding Interactions with the same affinity can have markedly different association and dissociation rates as seen in Figure 1 3 An antibody or small molecule that has a high affinity low K value for a protein target may be a poor drug in vivo if tt has a very high dissociation rate and thus can be easily displaced by another molecule This kind of information is easily obtained from an SPR experiment but would not be uncovered using a method such as isothermal calorimetry ITC that m
65. at the analyte binding site thiol coupling can be used to increase the ligand activity It is typically used when the ligand contains cysteine residues far from the analyte binding site Thiol coupling can be achieved on GLX chips by sequential injections of the reagents in Table 5 7 Table 5 7 Thiol coupling on GLX chip ligand surface is then regenerated and ready for the Injection Reagent Reaction to Chip Surface ligand analyte interaction analysis Figure 5 2B 1 EDC sulfo NHS Activation of carboxyl groups 2 Cysteine Creation of thiol groups 3 Aldehyde coupling if the ligand contains or sn os 3 anam Deactivation of carboxyl aldehyde groups not located at the analyte binding Tancian groups site aldehyde coupling can be used to increase DTNB 5 5 dithiobis ae ap l 4 i l Formation of disulfides the ligand activity It is typically used to increase 2 nitrobenzoic acid the activity of human antibody in which case the 3 Bead Immobilization of ligand by g i substitution of disulfides polysaccharide side chain of an antibody is oxidized by NalO sodium peroxide to create aldehyde groups Aldehyde coupling can be achieved on GLX chips by sequential injections of the reagents found in Table 5 6 A Capture surface method Capture reagent NA VA YA gt i i ii Surface OOOO O Surface _ ae E Analyte Q Q wa Y Y Y tnd NK YY Y ll ll ll ll ll Capture reagent V4 WH SSA Capture
66. ata table as shown in Figure 4 19 The report point created in another dataset may be imported to the current dataset by right clicking the data table and choosing Add Report Point 7 ProteOn XPR36 Experimental Design and Application Guide 3 w h e amp ma w w exe ee Se Se re mh i hw ie Tome Ca bercee mate sezeee om ue w e x om bed we un w a a a hod nua k z w agra m Fig 4 19 Report point values are shown in a new column of the data table 4 5 5 Data Presentation ProteOn Manager software presents the results of an interaction analysis as a data table The data table shows all the fitted parameters by default The parameter list can be changed by dragging the parameter columns in or out of a parameter database Column Chooser to customize the parameter list This option is available in the pop up menu by right clicking the data table The data table is automatically grouped in the same way as the sensorgram sets typically by ligand channels and may be ungrouped to display all the values as shown in Figure 4 20 In addition the data table panel can be resized by clicking the arrow buttons at the top left corner The values in the data table can be selected and copied to spreadsheets A 172 i 4 LIA 9 S0E 05 140 85 LSE 10 TAT Lia DI IL PESHT 2006 09 6196 05 9 50605 140 85 L610 12 55 tus vi IL P
67. ate Ligand Analyte Ligand lt Ligand poo poo 1 capture reagent 2 capture reagent Chip surface Analyte rabbit IgG Ab Ligand biotinylated goat anti rabbit IgG Ab 1st capture reagent histidine tagged streptavidin 2 capture reagent Tris NTA 1 capture reagent 2 capture reagent Chip surface Fig 3 32 Workflow of the regenerable biotin capture surface based on HTG and HTE chips 3 3 8 SPR MS Analysis SPR matrix assisted laser desorption ionization MALD MS coupling has been a constant demand in biology It combines the advantages of SPR and MS for analyte characterization and identification in a single experiment Traditionally the analytes are collected by elution from the sensor chip surface after kinetic analysis However it is possible to directly detect surface captured analytes on an SPR sensor chip with no elution steps since an SPR sensor chip is coated with a conductive gold layer that makes it highly adaptable to MALDI MS A new method is possible for SPR MALDI MS coupling using the ProteOn XPR36 system that allows direct detection from the chip surface Published Applications Proof of Concept for SPR MS Analysis Luo R et al 2012 Analyzing binding kinetics with surface plasmon resonance complemented with direct mass spectrometry on the same sensor chip BioRadiations Aug 2012 Roth S et al 2011 Secondary analysis of SPR based arrays by direct use in MALDI tim
68. ative control were immobilized in five ligand channels leaving one ligand channel blank to serve as reference Figure 3 35 A concentration series of each antigen was injected in six analyte channels Ligand surface regeneration was performed before each new analyte injection Response RU O 50 IL 2 cytokine IL 2 antibody A 2 E a q i Me Time min 200 Reference channel 150 50 or 100 fi Li ap Aa r h met airnean 5 Si PET NE EEEE a al P N 3 porate inahin ninian F amp 504 f a neis ii a 2 A Saneee ieee selene he 5 f E EE EEA Pee ES iE a tle Sh iaa PORN Te TER abe BT Se A ant op o _ O 2 4 6 8 10 12 Time min 50 IL 4 cytokine IL 4 antibody 0 2 4 6 8 10 12 Time min 200 Fig 3 35 Kinetic analysis for four Ag Ab pairs on a single sensor chip RU response units or a ie E O Q op 0 or O 2 4 6 8 10 12 Time min 99 ProteOn XPR36 Experimental Design and Application Guide 3 4 2 Biosimilar Assessment Antibody drugs have constituted the major part of biotherapeutics Biosimilars are generic versions of existing approved antibody drugs Determination of the Ab Fc receptor binding affinity of biosimilars is essential for understanding the mechanism by which the drug functions and the similarity between biosimilars and the corresponding original drugs The N linked oligosaccha
69. ay system is covered by Bio Rad patents including United States patent numbers 8 111 400 8 105 845 7 999 942 and 7 443 507 This product or portions thereof is manufactured and sold under license from GE Healthcare under United States patent numbers 5 492 840 5 554 541 5 965 456 7 736 587 and 8 021 626 and any international patents and patent applications claiming priority 115 Life Science Group Bulletin 6414 Rev B US EG Bio Rad Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 3065 7550 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 6965 Germany 089 31 8840 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 026674 55 05 Taiwan 886 2 2578 7189 Thailand 1800 88 22 88 United Kingdom 020 8328 2000 14 1372 0814 Sig 1213
70. blished and simultaneously analyzed with IL 1B was implemented The synergy of the two proteins was observed in the sensorgrams The synergy resulted in the fitting to the 1 1 Langmuir model while the single receptor showed more complicated binding mechanisms Figure 3 21 44 Applications Sensor chip Experimental Design R co R 1 0 R co R 1 10 Models 4 Response Ligand bound receptor exists in open and closed states in the absence of co receptor Time Fit with heterogeneous ligand binding model A Response Closed state of receptor stabilized by co receptor d Time Fit with simple 1 1 Langmuir binding model Binding Mechanism Sensorgrams with Fitting Fig 3 21 The experimental design binding mechanism and sensorgrams with fitting in the analysis between IL 1 IL 1 R1 and RAcP Characterization of the Interaction of Ubiquitin and Ubiquitin Binding Domains Marchese A 2011 Identification of a novel ubiquitin binding domain by SPR Bio Rad ProteOn Webinar Series Ubiquitin modification of proteins is involved in proteasomal degradation and also serves many non proteasomal functions in a wide variety of biological functions The functional outcome of ubiquitin modification is dependent upon noncovalent interactions with ubiquitin binding domains This webinar outlines SPR experiments in which a biotinylated peptide encompassing a novel ubiquitin bi
71. body interacting with small molecules on liposomes 2 small molecule drugs partitioning between aqueous solution and lipid bilayer and 3 membrane disruptive peptides interacting with liposomes Figure 3 28 The poster shows the value of using these products for lipid membrane and membrane orotein research Ligand Analyte Ligand Analyte j AA A gt FITC labeled Anti FITC Ab DSPC liposome Plain POPC liposome Melittin J Fluorescein group y BEE EFS EOS Fre Ligand Analyte lt cena i gt th x ns p gt At a low concentration At a higher concentration At an even higher melittin binds to the surface melittin forms pores that concentration melittin of the lipid membrane span the lipid membrane ruptures the liposome to Plain POPC Small molecule drug produce micellar structures liposome Fig 3 28 Use the lipid membrane protein application kits to analyze liposome protein liposome peptide and liposome small molecule interactions 49 ProteOn XPR36 Experimental Design and Application Guide A POPC DSPC CHOL NH SO 800 800 600 T T 2 2 Q 400 9 io op O O cr 200 O 4 O 100 O 100 200 300 400 100 O 100 200 300 400 Time sec Time sec Ketoprofen 1 amp Pindolol 2 E Tolmetin 3 E Sulpiride 4 E Naproxen 5 E Tetracaine 6 E Alprenolol 7 E Dibucaine 8 amp Metoprolol 9 M amp Hydroclorothiazide 10
72. c analysis with high accuracy and efficiency Table 3 2 The kinetic analysis could be used to derive the binding energetics and obtain the architecture of the binding interface 40 Applications Response RU Response RU Response RU TEM1 R243A S235A 250 ee S F a ee tag 150 ee ae os tite 100 4 AE T j iioii mau Pe ee eae ee ee ee EST ET Pe l 0 2 4 6 8 10 12 14 Time min TEM1 R243A S130A 250 200 150 J a D e y i Pe 100 ff ae i Bii E Fi A af i lane i ne ne Nee r 0 fF oe OO Lt E ae eee lf p a aaan A pn eii N aaa aaa l l 0 2 4 6 8 10 12 14 Time min TEM1 S130A S235A 250 200 ee w AAA he ee 150 ra gt i E o ma y BO me ay i Pa aaa a i H yt ae n Bia iiia TPE ae a 50 ae a Ay s an EE Nr red teas Wiican pte ac os hee pe ee N ad o eer 0 2 4 6 8 10 12 14 Time min TEM1 K234A 250 D oO o N a O Q ep oO or O 2 4 6 8 10 12 14 Time min TEM1 E104A 250 200 4 Y gA Bi g 150 _ g j Pi 5 2 100 Fi iii AA pen AO O 2 4 6 8 10 12 14 Time min Fig 3 16 Kinetic analysis for the interactions between BLIP and five TEM1 mutant proteins Each set of six sensorgrams displays the responses from the six BLIP analyte concentrations 600 nM 300 nM 150 nM 75 nM 37 5 nM 18 75 nM inter
73. ce should be determined The desired immobilization level is calculated using the following equation Rmax IS the desired maximum response when the ligand interacts with an analyte M is the molecular weight of the analyte M is the molecular weight of the ligand and n is the stoichiometric coefficient of the interaction the analyte ligand ratio For kinetic analysis aiming for an analyte response with R may lt 200 RU is recommended The approximate capacity of the amine coupling GLX GLC GLM and GLH chips the biotin capture NLC chip and the histidine tag capture HTX HTG and HTE chips is as follows GLC 8kRU GLM 12kRU GLH 20 KRU NLC 2 KRU HIG SkRU HTE 12 KRU Optimizing Immobilization Conditions In the ProteOn XPR36 system the experimental conditions can be conveniently optimized by injecting reagents across multiple channels with each channel having different conditions for example varied concentrations of ligand and analyte GLX Sensor Chips Activation When using the amine coupling or GLX chips mix 1 ethyl 3 8 dimethylaminopropyl carbodiimide hydrochloride EDC and N hydroxysulfosuccinimide Sulfo NHS to produce the activation solution The activation reagents are typically prepared as a mixture of 1x EDC sulfo NHS and should be mixed immediately before the injection It is recommended to use a contact time of 60 sec for moderate activation and 300 sec for high activation levels For the GLH chip
74. centration to up to 500 mM in the analyte Increase the buffer salt buffer and running buffer using NaCl for example to shield the S k concentration electrostatic charges It is important to verify that the high salt concentration does not affect the ligand or analyte activity Increase the pH of the analyte buffer and running buffer to EEEE bun reduce the positive charges contributing to electrostatic NSB It z N p is important to verify that the high pH does not affect the ligand or analyte activity Add 0 05 Tween 20 and or 0 1 BSA to the running buffer to Add 0 05 Tween 20 and reduce both electrostatic and non electrostatic NSB 0 1 BSA f or 0 1 BSA may also be used to saturate the chip surface to block potential NSB sites Create an appropriate reference surface by capturing a reference protein unrelated to the ligand such as BSA to the same level as the ligand The reference protein does not reduce NSB directly e but it shields the charges on the chip surface as much as the ligand NSB will be corrected for by subtracting the reference Create an appropriate reference surface When using a complex analyte sample like serum or crude lysate dilute the sample with the running buffer a five to Eee tE tenfold dilution is usually recommended Higher dilution rates i 5 should be used if the sample is very concentrated Use simple prepurification methods to remove the majority of the A Vse prepunfication cont
75. ction 2D oO C C Time g m gt J4 g Reference Subtracted a Analyte Injection Time Option 1 Channel reference Ligand channels Il l l l l l Raw Data Analyte Injection p Hf ff HF HF FE oO S E i E E C g ee E E E O i m ij amp O E EEEE Reference Subtracted Fal Analyte Injection RU Time Option 2 Interspot reference Fig 4 9 The two blank surface referencing options in the ProteOn XPR36 system RU response units Blank buffer referencing also known as double referencing is performed on a ligand surface with a blank buffer either a running buffer or a negative control sample flowing over it The reference responses are collected on ligand surfaces during a blank buffer injection that is blank buffer reference ligand surface blank buffer as shown in Figure 4 10 Blank buffer referencing is used to correct for baseline drift resulting from the changes of the ligand surface Ligand Surface Analyte Injection Analyte Interaction surface Ligand Surface Blank Injection wnt YF NF NZ Reference surface Fig 4 10 Schematic diagram of a blank buffer reference The ProteOn XPR36 system offers two blank buffer referencing options as illustrated in Figure 4 11 Injection referencing is the reference method traditionally used in commercial SPR biosensors It requires a blank buffer injection pe
76. ction by simultaneously injecting a full concentration series of an analyte without having to regenerate the ligand surface in contrast to traditional SPR instruments that allow only sequential injections In addition the ProteOn sensor chip can be set up with 36 different target proteins for the acquisition of information rich kinetic screening data in a high throughput fashion This provides a novel method for screening analytes simultaneously against 36 ligands further increasing the throughput of kinetic screening and enabling a rapid comparison between many interactions Advantages include Efficient experimental optimization Accurate kinetic analysis Compatible with crude samples High throughput screening 30 Applications Published Applications One shot Kinetics approach to monitor the interaction One Shot Kinetics Approach for Antibody Screening between multiple concentrations of the analyte IL 2 and a aa eon aaa a aain the igand antil 2 antibody immobilized with multiple Bio Rad Bulletin 3172 conditions in a single analyte injection Figure 3 2 The 6 x 6 interaction array is used to generate 36 sensorgrams simultaneously It not only increases throughput but also provides novel referencing options This technical note Bronner et al 2005 employs the ProteOn XPR36 system to determine the kinetics of the interaction between interleukin 2 IL 2 and anti lL 2 antibody The experiment was performed using the
77. d experiment files C Click Save to start the export process When completed click Close The exported file has the extension name pomexp and it can be imported only to ProteOn Manager software Note It is possible to export the data in an experiment file in a spreadsheet format Please refer to the user manual for details 2 Import experiment files A In the menu bar click File select Import and choose Experiment Protocol File in the submenu to open the file browser Select the experiment file to import Hold the Ctrl key to select multiple experiment files B Click Open to start the import process When the import process is completed click Close to close the database browser The imported experiment file is added to the database Note If the Import and Export options are grayed out check that the ProteOn Manager USB key is in place NeutrAvidin is a trademark of Thermo Fisher Scientific Inc Excel is a trademark of Microsoft Corporation Integral Molecular is a trademark of Integral Molecular Inc MemLAYER is a trademark of Layerlab AB Tween is a trademark of ICI Americas Inc For technical support call your local Bio Rad office or in the U S call 1 800 424 6723 This product is for research use only 2014 Bio Rad Laboratories Inc Reproduction in any form either print or electronic is prohibited without written permission of Bio Rad Laboratories Inc The ProteOn XPR36 protein interaction arr
78. del The bivalent analyte model is used when an analyte has two separate binding sites The following equation describes binding of a bivalent analyte Kar Kao A B AB B ABB Kj Kyo Equation 5 where A is the analyte and B is the ligand The association and dissociation of the first binding event is described by k and k respectively while k and k respectively describe the association and dissociation of the second binding event The first event will yield a traditional 1 1 kinetic fit where the second binding event will cause the ligand analyte complex to stabilize thus changing the kinetics of the reaction Therefore a sensorgram of a bivalent analyte binding to ligand is the result of two separate kinetic processes occurring in tandem Heterogeneous Analyte Model When an analyte is heterogeneous analyte may bind to the ligand in two different locations This can occur naturally ifa sample is not completely pure or if there are two different types of analyte in solution Thus a sensorgram of a heterogeneous analyte binding to immobilized ligand represents the sum of two separate binding interactions If one analyte has a naturally higher affinity than the other analyte the two may compete for binding of the ligand and the sensorgram data will reflect the binding kinetics of the higher affinity ligand The following equations are used to describe and model the binding of a heterogeneous analyte k k at a2 A1
79. e bulk analyte concentration as shown in Equation 10 The concentration of an unknown sample is calculated by comparing the binding response under these conditions to a standard curve of binding responses for known concentrations as shown in Figure 4 18 Initial Binding Rate k A Equation 10 1 200 1 000 4 XT 800 fa 2 _ 600 sae O pe 400 a 200 ee l _ A f 40 20 0 20 40 60 80 100 120 Time sec Fig 4 18 The mechanism of concentration analysis k is the mass transport coefficient in the unit of RU Ms In ProteOn Manager software the standard four parameter logistic equation is employed to determine the unknown concentration Note that the concentration analysis again presents the scopes of fitted or constant and global or grouped for parameter setting The definitions are the same as those described in the previous section 4 5 4 Report Point A report point is created to directly read the average value of sensorgrams within a specified time range Sensorgram fitting is not involved in this procedure A report point is often used to measure the immobilization level of ligands or qualitatively compare the responses with different analyte injections A report point is created in two steps 1 right click and drag to select a time range and 2 right click the selected time range to create a report point The report point values are shown in a new column of the d
80. e Langmuir model is used select Simultaneous k k in the Model Options box for full sensorgram fitting or Off Rate Analysis for dissociation fitting Click Next CONCENTRATION Select Analysis and choose Concentration in the submenus Select type Set parameter Review result v 2 o D Cc ca Cc gt 2 l Q C Good SPR Inspect the displayed regions selected for analysis and the settings of analysis parameters Click Next to analyze the sensorgrams The calculated SPR results are displayed in the report table Click Finish to save the fitted sensorgram set under the dataset NO result SPR result Troubleshoot with the data processing steps 85 ProteOn XPR36 Experimental Design and Application Guide 4 8 Options for Dataset Export i File View Background Taree ainerent ways of exporting a dataset are explained Lip Page Setup IDALA ioe i in the following GP Print amp Print Option 1 Print a ProteOn Manager Report i Export Document Y PDFFie OnT XPR36 Kinetic Repor d Send via E Mail gt ile 7 Se 1 In the Navigator sidebar enter the Analysis Dataset 00 M tab and choose a dataset When a graph with RIF Fie IL2 i i i ile L1 Anti IL2 Ah Acetate pH 4 5 overlaid fitted curves is preferred select an analyzed ms Maciek Wace wens
81. e Tagged Proteins The HTG and HTE sensor chips feature a novel tris NTA 3 x NTA complex for improved capture of histidine tagged proteins This tris NTA complex has a significantly higher binding stability than the traditional NTA so minimal ligand leaches off the surface and the sensorgram NLC Sensor Chip Immobilization of Biotinylated Ligands The NLC sensor chip is functionalized with NeutrAvidin bound to the alginate polymer and can capture biotinylated proteins peptides and nucleic acids It can capture 2 000 RU of IgG or 500 RU of DNA The NLC baseline remains stable NTA is the traditional method sensor chip is ideal for immobilizing ligands without used to capture histidine tagged proteins but the binding amine coupling but requires that the ligand be modified is less tight causes ligand to leach off the surface and with biotin prior to immobilization Figure 2 6 results in unstable baselines and distorted kinetic results all of which can lead to an inaccurate fit to a binding A model The tris NTA complex contains three NTA moieties for improved binding stability and increased binding selectivity to histidine tagged proteins Figure 2 7A The as eas G05 OSS oS oO NeutrAvicin tris NTA complex is attached to the alginate polymer e amp matrix on the sensor chip and is activated by injecting nickel Il ions In order to achieve optimal performance Incident light Detector in various applications the surface dens
82. e molecular weight of the ligand and n is the stoichiometric number of the interaction analyte ligand For high quality kinetic analysis it is recommended to aim for analyte response with Rmax lt 200 RU The approximate capacity of the amine coupling chips GLX GLC GLM and GLH the biotin capture chip NLC chip and the histidine tag capture chips HTX HTG and HTE are listed as follows GLC 8 kRU GLM 12 KRU GLH 20 kRU NLC 2 KRU HTG 5 kRU HTE 12 KRU Optimize the immobilization conditions in a ProteOn XPR36 system immobilization conditions are optimized by injecting ligand across multiple channels with each channel containing the ligand at a different condition such as concentrations or pH Kinetic analysis is performed for all ligand channels at once 112 Quick Guides For amine coupling chips GLC GLM and GLH chips typically activation reagents for amine coupling chips are used at concentrations of 20 mM 1 ethyl 3 6 dimethylaminopropy carbodiimide hydrochloride EDC and 5 mM N hydroxysulfosuccinimide Sulfo NHS but could be diluted in distilled water when a low immobilization level is needed Activation reagents should be mixed immediately prior to injection Contact time is 60 sec for moderate activation and 300 sec for high activation levels Usually the ligand is prepared in the concentration range of 0 5 25 ug ml Ligand coupling buffer is typically 1 pH unit lower than the liga
83. e of flight mass spectrometer Poster presented at HUPO World Congress Geneva Switzerland Sep 2011 This article and poster describe the proof of concepts of SPR MS analysis using the ProteOn XPR36 system In the experiment B amyloid peptide fragments were first analyzed and captured by SPR and subsequently analyzed by MALDI MS to identify the mass of each individual peptide fragment For kinetic characterization One shot Kinetic analysis of the interaction of 6E10 Ab and B amyloid 1 40 was performed The surface was regenerated with phosphoric acid and reloaded with B amyloid 1 40 80 nM for MS analysis The chip was rinsed in water and mounted to a customized adaptor and then MALDI MS analysis was carried out in amass spectrometer Figure 3 33 53 ProteOn XPR36 Experimental Design and Application Guide Desalt Array Insert Array in Adapter 50 po Oo a0 PONR AAAA Ly n k 2 30 D yY aT Ayyy s a 20 fA VE DWT POV PU WO eo 0 AI 10 O ANA x 10 OT T T T TT T TT TT T T T E 200 100 O 100 200 300 400 500 600 700 800 900 1 000 Time sec Insert Adapter in Holder Add Matrix Read in Mass Spec 1 500 ity i 1 000 Intens 500 LAN ah eee at FP na At Aap aa iyagi gla tint nd dtl ag wh es a 500 1 000 1 500 2 000 2 500 3 000 3 500 4 000 4 500 5 000 m z Fig 3 33 SPR MS analysis using direct MALDI MS analysis from the chip surface
84. e regions used to calculate the constants are shown in the graph Figure 6 1 k and ky Equilibrium Ky Association l _ Dissociation Response RU Baseline Regeneration Time sec Fig 6 1 Descriptors of an SPR sensorgram and regions to calculate the kinetic and equilibrium constants RU response units In an SPR sensorgram which parameters are necessary for the calculation of kinetic constants For the association kinetic constant k the analyte concentration is necessary for the calculation For the dissociation kinetic constant k no parameter is needed 6 3 Sensor Chips What types of sensor chips does Bio Rad offer ProteOn GLC sensor chip for general amine coupling polymer matrix layer with compact binding capacity of approximately one protein layer ProteOn GLM sensor chip for general amine coupling polymer matrix layer with intermediate binding Capacity ProteOn GLH sensor chip for general amine coupling polymer matrix layer with high binding Capacity ProteOn NLC sensor chip for capturing biotinylated molecules polymer matrix layer containing NeutrAvidin with compact binding capacity ProteOn HTG sensor chip for capturing histidine tagged proteins polymer matrix layer containing tris NTA complexes with compact binding capacity ProteOn HTE sensor chip for capturing histidine tagged proteins polymer matrix layer containing tris NTA complexes
85. e two following approaches 1 Set two consecutive ligand injection steps with a pause step in between The first ligand injection step is short and measures the ligand immobilization rate During the pause step you may fine tune the second ligand injection according to the ligand immobilization rate determined in the first step to achieve the desired Immobilization level 2 Seta single long ligand injection step and monitor the ligand immobilization process When the desired immobilization level is reached press the Abort button to end the ligand injection step Deactivation Deactivation uses 1 M ethanolamine HCI at pH 8 5 to block any remaining activated carboxyl group on the Chip surface It is performed in the vertical direction the same direction as the activation and ligand immobilization injections 92 Tips and Techniques NLC Sensor Chip Ligand Capture It is recommended to prepare the ligand in a concentration range of 0 5 ug ml to 25 ug ml Typically using a slow flow rate of 30 ul min is suggested to reduce ligand use during ligand injection the contact time may vary from 1 min to over 10 min depending on the immobilization level needed These conditions provide a starting point for further optimization of experimental conditions Note The NLC chip does not need activation and deactivation with biotin is optional HTX Sensor Chips Activation and Ligand Capture Refer to the product insert part numb
86. eases as analyte is flowed over the sensor chip and associates with the immobilized ligand and then decreases as the analyte solution is replaced with buffer and the binding complex dissociates If binding equilibrium is reached during the association phase the sensorgram will reach a constant plateau before the analyte solution is replaced with buffer and the binding complex dissociates Fitting the sensorgram data to a binding model allows for the calculation of the association and dissociation rate constants and determination of the binding affinity Traditionally kinetic measurements with SPR usually involve sequential injections of analyte at increasing concentrations over the same ligand surface which requires complete removal of the analyte or regeneration of the ligand surface between analyte injections In an ideal case regeneration of the ligand surface is observed in the sensorgram as a sharp response change after dissociation to restore the baseline to the original level Regeneration is usually done with a combination of dilute surfactants salts and acids or bases however care must be taken during regeneration to avoid denaturing the immobilized ligand or removing ligand from the sensor chip The ProteOn XPR36 system characterizes the following aspects of a biomolecular interaction Specificity of the interaction Rate of the interaction k Stability of the complex k Strength of the interaction Kp Kp
87. easures binding affinity based on binding at equilibrium In addition knowing the kinetics of a small molecule interaction allows for more accurate analysis of quantitative structure activity relationships as different structures can be evaluated by their separate effects on association and dissociation as opposed to affinity alone Association Dissociation K 1 0 nM 804 A 10 nM k 1 M tsec Faer 1x10 1x10 1x104 10 gt 1x105 1x 10 1 10 1x10 1x10 1x102 Q O Response RU D O O 200 400 600 800 1 000 1 200 1 400 Time sec Fig 1 3 Sensorgram plots showing the response in RU versus time for five different interactions with the same affinity K 1 0 nM but markedly different association k and dissociation k rate constants A antibody 000000 e0e000 Evaluate binding against 6 analytes such as small inhibitor molecules Figure 1 4 The experimental workflow of the 6 x 6 interaction array 12 ProteOn XPR36 Technology ProteOn Manager software gives you the option of 1 4 Advantages of the using seven different binding models to analyze your 6 X 6 Interaction Array sensorgram data When running an experiment using the ProteOn XPR36 system the 6 x 6 interaction is formed The experimental workflow is shown in Figure 1 4 Langmuir simple 1 1 bimolecular interaction Simultaneous fitting of k and ky Fitti
88. echanism of action Confirmation of biomolecule binding to a target Screening of fragment libraries Validation of IC EC values during hit to lead optimization Characterization of immune responses Step 1 Immobilize up to 6 ligands in the vertical direction Fig 1 2 The ProteOn 6 x 6 interaction array on a sensor chip Step 2 Inject up to 6 analytes in the horizontal direction Step 3 Detail showing one of 36 interaction spots 11 ProteOn XPR36 Experimental Design and Application Guide Some classic applications of the ProteOn XPR36 system are in antibody engineering Epitopes on an antigen can be characterized by epitope mapping a process by which the affinities of an antibody to site directed mutants of a single antigen help pinpoint the location of an epitope An investigation of the epitope specificity or epitope binding of different antibodies can be done on the ProteOn XPR36 system using the sandwich assay In this assay a second antibody is injected over a previously formed antigen antibody complex to see whether or not the second antibody can still bind Binding of the second antibody to the antigen antibody complex or the formation of a sandwich is an indication that the second antibody recognizes a different epitope than the first antibody SPR can also be used to determine the active concentration of an analyte in a crude or impure sample by probing the sample of interest under m
89. echanism of cytokinesis A set of tools were used to study the connection between mitotic spindle and plasma membrane during cytokinesis It was discovered that the C1 domain of the centralspindlin subunit MgcRacGAP associated with the plasma membrane by interacting with polyanionic phosphoinositide lipids The ProteOn XPR36 system was used in the analysis of the binding of MgcRacGAP C1 domain to phosphatidylethanolamine vesicles containing 5 PtdIns 4 P or Ptdins 4 5 P The model of MgcRacGAP C1 domain membrane interaction was established Figure 3 20 A gt Mixed phospholipid MgcRacGAP C1 liposome domain B Phosphoinositide Fig 3 20 Analysis of the binding of MgcRacGAP C1 domain to phosphatidylethanolamine vesicles containing 5 phosphatidylinositol 4 phosphate PtdIns 4 P or phosphatidylinositol 4 5 bisphosphate PtdIins 4 5 P Characterization of the Interaction of IL 1 R1 and IL 18 in the Presence of RAcP Issafras H 2011 Antibody characterization using the ProteOn XPR36 system a multiplexed SPR biosensor Bio Rad ProteOn Webinar Series This webinar reports the work that investigated the binding of interleukin 18 IL 18 and the receptor IL 1 R1 in the presence of the receptor accessory protein RAcP Issafras 2011 Utilizing the parallel channel experimental configuration in the ProteOn XPR36 system interaction surfaces with different mixing ratios of the two proteins and reference surfaces were esta
90. ection step can cancel out these ERIR pe differences and lead to more reliable binding results To Run an Experiment With a Highly Q Refractive Cosolvent DMSO DMSO 4 8m 6m 4m Om 6 2ml 4ml 6ml 10m 1 Flush the instrument with ligand immobilization buffer in buffer position B The ligand immobilization buffer Fig 4 26 Preparation of DMSO dilutions for EV calibration Prepare usually does not contain cosolvent unless it is known two dilutions of DMSO in fresh running buffer one above and one below s a the concentration used for the DMSO running buffer In this example not to interfere with immobilization the running buffer contains 5 DMSO Mix the two dilutions at the 2 Immobilize the ligand onto the sensor chip ratios described in the diagram to create a concentration series that has concentrations that cross over the DMSO concentration in the running 3 Determine the cosolvent concentration to be used in buffer the experiment for example DMSO 5 that will keep the analyte soluble DMSO concentrations up to 10 are acceptable 4 Prepare an analyte stock solution EV calibration standards and running buffer These solutions should be prepared similarly to make the EV correction EVC the most accurate 81 ProteOn XPR36 Experimental Design and Application Guide 7 In the Protocol tab after creating your protocol click and drag the EV correction step group to the end of your protocol The EV correction step
91. ed on a self assembled monolayer Figure 2 9 It is designed to be used with the ProteOn LCP capturing reagent kit for lipid protein lipid small molecule and membrane protein protein interaction analysis The reagent kit activates the chip surface by a biotinylated DNA tag so that the chip is able to capture DNA labeled lipid assemblies through DNA hybridization The reagent kit attaches DNA tags to the lipid assemblies in order to anchor them to the chip surface It is possible to capture two or more layers of lipid assemblies for additional sensitivity This method of capture allows for lipid based interaction analysis including the analysis of membrane proteins embedded in a lipid bilayer Figure 2 10 The LCP sensor chip provides a novel hydrophilic surface chemistry for capturing lipid assemblies which allows for novel lipid based applications Bio Rad offers the liposome capturing kit as an all in one kit composed of the LCP sensor chip and the LCP capturing reagent kit The advantages of this application kit are summarized below Hydrophilic surface chemistry Low nonspecific binding Multiple layer capture capability High regeneration capability High throughput A Analyte NeutrAvidin Incident light Detector S eo eo ee Kinetic _ _ LCP chip LCP chip Hybridization of the Biotin ssDNA two DNA strands Biotin ssDNA Chol dsDNA 1 liposome Fig 2 9 ProteOn LCP sensor chip A planar
92. ed to a kinetic model using a mathematical algorithm In ProteOn Manager software the user may choose among seven different binding models with which to perform the interaction analysis However it is recommended that SPR interactions are fitted to the simplest model possible Binding Models Langmuir The most commonly used binding model for SPR biosensors is the Langmuir model It describes a 1 1 interaction in which one ligand molecule interacts with one analyte molecule In theory the formation of the ligand analyte complex follows second order kinetics However because the majority of SPR biosensors are fluidics based and capable of maintaining a constant analyte concentration in a continuous liquid flow complex formation actually follows pseudo first order kinetics In addition this model assumes that the binding reactions are equivalent and independent at all binding sites It is also assumed that the reaction rate is not limited by mass transport Many interactions adhere to this model in which the interaction is described by the simple equation shown below where B represents the ligand and A is the analyte The rate of complex formation is represented by the association constant k in the unit of Mts and the rate of complex decay is represented by the dissociation constant k in the unit of s as given by Equation 1 Association Phase Ka gt 1 Ka A B AB Equation 1 In a kinetic analysis the
93. ed to correct for baseline drift resulting from the changes of the ligand surface Analyte Interaction surface Blank Surface Analyte Injection The novel 6 x 6 experimental configuration in the ProteOn XPR36 system offers a comprehensive set of aed Seat malas ge Ba referencing options The referencing options canbe pes selected from the Process menu as shown in Figure 4 7 The available referencing options are listed below Q Q Analyte One Shot Kinetics Experiment ProteOn Manager Pp g File Edit Yiew Process Analysis Tools Help m a 2 Channel Reference gt Interspot v Double Reference gt Column b ball Navigator Auto Process Row b Reference surface Instrumet Protoco Injection Alignment gt Correct for Excluded volume Run Baseline Alignment gt Fig 4 8 Schematic diagram of a blank surface reference Data Artifact Removal r Manual Alignment y gt fe HH amp The ProteOn XPR36 system offers two blank surface referencing options as illustrated in Figure 4 9 Remove Processing Panel Type Channel referencing is the reference method traditionally used in commercial SPR biosensors It involves reserving a portion of the potential interaction surfaces for use as blank surfaces H One Shot Kinetics Experiment ProteOn Manager File Edit view Process Analysis Tools Help
94. ee amine group on the ligand molecule to the chip surface the ligand activity is usually not very high 94 Tips and Techniques If low ligand activity is caused by incorrect ligand Table 5 6 Aldehyde coupling on GLX chip orientation in amine coupling there are multiple Injection Reagent Reaction to Chip Surface alternative ligand immobilization methods typically used EDC sulfo NAS Activation of carboxyl groups as solutions 2 Carbohydrazide Creation of amine groups 3 ST RANG OnE Deactivation of carboxyl 1 Capture surface use a chip surface functionalized groups with some capture agent such as antibody biotin 4 Ligand Immobilization of ligand by Schiff base reaction binding proteins avidin family proteins histidine tag binding reagents etc It should be noted that the ligand must be biotinylated or histidine tagged to use biotin or histidine tag binding surface chemistry Figure 5 2A Protected immobilization premix the ligand with a known reagent binding to the analyte binding site for example premix kinase ligand with a Known inhibitor in the case of screening new inhibitive compounds The ligand reagent complex formed in the solution guarantees the right orientation of the ligand when it is immobilized on the chip surface The Stabilization of ligand by reduction of Schiff base NaB CN H sodium cyanoborohydride Thiol coupling if the ligand contains thiol groups not located
95. ensor chip TEM1 was immobilized to approximately 1 500 RU and BLIP was injected in a twofold dilution series ranging from 600 38 nM RU response units GLH Sensor Chip High Binding Capacity The GLH sensor chip is designed with a high density alginate polymer that contains an increased number of carboxylic acid groups to amine coupling ligands at a very high gt 20 kRU surface capacity This dense alginate layer on the GLH chip is far superior at binding high capacity ligand surfaces when compared to results of ligand immobilizations done using the GLC and GLM chips Figure 2 4 This sensor chip is ideal for probing orotein small molecule lt 1000 Da interactions as the high capacity surface gives an increased binding response A comparison between the GLH sensor chip and a competitor s high capacity sensor chip shows the full advantage of the ProteOn chip s easily activated carboxylic groups rendering significantly higher binding capacity and activity and thus much higher analyte response Figure 2 5 and Table 2 1 A Analyte Incident light Detector Response RU Time min Fig 2 4 ProteOn GLH sensor chip A the dense alginate coating on the GLH sensor chip responsible for creating a high capacity surface B sensorgrams of the interaction between the carbonic anhydrase Il 30 kD and the inhibitor 4 carboxybenzenesulfonamide CBS 201 Da using the GLH sensor chip Carbonic anhydra
96. ensorgrams can be adjusted in the Data Grouping screen and individual sensorgrams can be removed from or added back to the sensorgram display in the Interaction screen The sensorgram graphs can be selected or deselected by clicking their blue title bars In the menu bar click View and Select All Graphs to select all the sensorgram graphs While Auto data processing activities apply to all the sensorgram graphs Selected data processing activities apply only to the selected sensorgram graphs 68 Experimental Design 4 4 3 Sensorgram Processing Align the Sensorgram Set To perform reliable kinetic analysis a set of dose response sensorgrams for the same interaction are typically analyzed together to minimize system deviations This action requires the alignment of the graphs in a sensorgram set in both the vertical and horizontal dimensions In scientific experiments it is common to perform diagram or sensorgram alignment when processing multiple data groups in a dataset The alignment action for SPR sensorgrams is similar to those performed for other bioanalytical technologies such as spectroscopy ProteOn Manager software offers automatic injection alignment along the x axis and baseline alignment along the y axis available in the Process menu as shown in Figure 4 5 Injection alignment adjusts all the sensorgrams to share the same starting point along the x axis thus removing time differences among the different sensor
97. ent light Fig 2 7 ProteOn HTG and HTE sensor chips A structure of surface bound NTA molecule left and tris NTA molecule right Each individual NTA group is circled B alginate coating modified with compact density tris NTA on the HTG sensor chip C alginate coating modified with high density tris NTA on the HTE sensor chip A 300 4 D or a 200 4 cp Cc O Q ee ee a 100 idi 0 I I l I l I 200 100 O 100 200 300 400 500 600 700 800 Time sec J or oO ep O Q N X Time sec Fig 2 8 ProteOn HTG and HTE sensor chips A sensorgrams of the interaction between the histidine tagged protein A and human IgG showing the ability of the HTG sensor chip to resolve high affinity kinetics requiring long dissociation times Protein A was captured to approximately 60 RU and human IgG was injected in a twofold dilution series ranging from 100 nM to 6 3 nM B sensorgrams of the interaction between histidine tagged Erk2 an MAP kinase and the inhibitor Purvalanol B 432 9 Da showing that small molecules can be screened using the HTE sensor chip Erk2 was captured to approximately 12 800 RU and Purvalanol B was injected in a threefold dilution series ranging from 50 0 62 uM RU response units 2 3 3 ProteOn Sensor Chips for Capturing Lipid Assemblies Modified GLC and LCP Biological research focused on biomolecular interactions involving lipid assembl
98. equilibrium constant Kp in the unit of M is calculated from the two kinetic constants through the defining relation Kp Ky Ka Relating the interaction state to the SPR sensorgram is accomplished by applying specific equations relevant to the different sensorgram phases as illustrated in Figure 4 12 R Al Association R Roex IA alka A a K A Equation 2 Di iation k t issociation R Roe Equation 3 Equation 2 Equation 3 PP as A R 1 eka A kat R Re t Kp A F Association Dissociation Baseline Time Fig 4 12 An idealized sensorgram showing the baseline association and dissociation phases Analysis of the sensorgram curve in the association phase in which binding is measured while the analyte solution flows over the ligand surface allows the determination of the rate of complex formation There is an associated increase in response units over time as the complex forms on the chip surface Figure 4 13 outlines the derivation of Equation 2 A constant AB a R B B na AB B nax Ol R nax d AB _ dR _ odt k A B k AB dt KIAR ia R E KaR Rinax IA R 4 gk lA kat K IAl J Determines the Determines the time equilibrium level to reach equilibrum Fig 4 13 The derivation of Equation 2 13 ProteOn XPR36 Experimental Design and Application Guide As can be inferred from this derivation the c
99. er 10021524 included with the HTG and HTE reagent kit for details on how to use this kit When using the histidine tag capture chips or HTX chips it is recommended to prepare the ligand in a concentration range of 0 5 ug ml to 25 ug ml and using a slow flow rate of 30 ul min for the ligand injection to reduce ligand use The contact time may vary from 1 min to over 10 min depending on the immobilization level needed These conditions provide a starting point for the further optimization of experimental conditions Perform ligand injection immediately after the activation step to avoid nickel Il ion leakage and consequently a reduced immobilization level Table 5 2 HTX chip activation and ligand capture parameters Flow Rate Injection Reagent Orientation Volume ul ul min 1 10 mM NiSO Vertical 60 30 2 0 5 25 ug ml ligand Vertical Flexible 30 The ProteOn HTG and HTE chips are designed to capture histidine tagged proteins directly from crude media and purified proteins When capturing ligand from crude samples dilute the ligand sample before the capture to reduce nonspecific binding It is recommended to perform a significant dilution for example by 100 fold depending on the amount of active ligand in the sample Note The HTG and HTE chips require activation but not deactivation Ligand injection should be performed immediately after the activation step to avoid nickel Il ion leakage and consequently a reduced bind
100. ew The ProteOn XPR36 system uses the 6 x 6 interaction array of the ProteOn sensor chips ProteOn sensor chips contain more than just a gold layer they are coated with a modified alginate polymer that provides a solution like biomimetic environment for ligand immobilization The general use ProteOn GLC GLM and GLH chips are functionalized with carboxyl groups that react with surface exposed amines on the ligand tethering the ligand to the chip surface in a random orientation The ProteOn NLC chip is coated with NeutrAvidin for immobilization of biotinylated ligands and the HTG and HTE chips feature a tris nitrilotriacetate tris NTA or 3 x NTA surface for immobilization of histidine tagged proteins The respective chips can be used to capture a ligand at a site specific location The ProteOn LCP chip is used with the LCP capturing reagent kit for liposome capture Although choosing a suitable sensor chip for a particular interaction requires some research and planning this is time well spent considering the high quality data that can be obtained with the right sensor chip 2 2 ProteOn Sensor Chip Surface Chemistry ProteOn sensor chips are built with an alginate polymer matrix bound to a thin gold film on a sensor prism The alginate matrix can be functionalized with several different reactive groups to facilitate different immobilization surface chemistries The hydrophilic nature of the alginate layer creates a solution like en
101. for a detailed explanation of z this calibration and experimental guidance Time 4 4 5 Quality Standards for Processed Option 1 Injection reference Sensorgrams Ligand channels The following standards are used to judge the quality of processed sensorgrams Blank Surface Reference Subtracted 1 Processed sensorgrams the sensorgrams are LILILLILI E a a a a n ore ee aligned in both dimensions and artifacts such as nf Hf 5 air bubbles are removed This processing requires Baan LF a ES a E both good quality raw sensorgrams and appropriate Ead T software functions 2 ad A _ mk F 2 Good choice of referencing both blank surface amp d ouble Reference _ Bante Sensorgram referencing channel referencing and blank buffer referencing double referencing are appropriately performed The experimental design must ensure the incorporation of the correct referencing options ae The referenced sensorgrams should not show bulk Option Reaime rekone effects or baseline drift Although it is not required the best practice is to have no response jump Fig 4 11 The two blank buffer referencing options in the ProteOn XPR36 Present between the end of the association and the system RU response units beginning of the dissociation phases RU 3 Sufficient interaction time the interaction time or the time of analyte injection in the association phase is long enough to show curvature and t
102. g the capabilities of SPR technology the ProteOn XPR36 system maximizes the power of the unique 6 x 6 experimental configuration to combine concentration and kinetic analysis in a single experiment this is the so called quantikinetics workflow This new workflow will significantly enhance the efficiency and throughput in antibody production Published Applications Analysis of Sample Quantitation and Kinetics in a Single Experiment Ross G 2012 Antibody quantitation and full kinetic analysis in a single 45 minute experiment using the ProteOn XPR36 system Bio Rad ProteOn Webinar Series Ross G et al 2013a ProteOn XPR36 Quantikinetics antibody concentration and detailed kinetic analysis in a single experimental cycle Bio Rad Bulletin 6411 Ross G et al 2013b ProteOn XPR86 Quantikinetics antibody concentration and detailed kinetic analysis in a single experimental cycle Poster presented at Antibody Engineering amp Therapeutics Huntington Beach USA Dec 2013 This article Ross et al 2013a poster Ross 2013b and webinar Ross 2012 describe how concentration analysis is performed using an SPR biosensor and how quantikinetics is realized with the ProteOn XPR36 system Quantikinetics refers to the combination of sample quantitation and kinetic analysis in a single experiment to enhance efficiency and throughput in antibody production Figure 3 8 For proof of principle both purified antibody samples and supernatan
103. ged Protein Analysis The ProteOn XPR36 system combined with the HTG and HTE chips offers the capability to achieve high quality interaction analysis of histidine tagged proteins The HTG and HTE chips feature a novel Tris NTA technology that provides the binding stability and binding selectivity to histidine tagged proteins and surface regenerability of the sensor chips In the field of label free biomolecular interaction analysis this is a significant advance from the traditional approaches which exhibit considerable baseline drift due to low binding stability Benefits of HTG and HTE chips include Stability of histidine tagged protein capture for accurate analysis Selectivity for efficient online purification process Regeneration capability for low cost Published Applications A Wide Range of Applications for the HTG and HTE Sensor Chips Bronner V et al 2012 The ProteOn HTE sensor chip novel surface for stable capture of histidine tagged proteins for protein small molecule interaction analysis Bio Rad Bulletin 6254 Luo R et al 2012 Label free drug screening against histidine tagged proteins using novel ProteOn sensor chips Poster presented at Drug Discovery Chemistry San Diego USA Apr 2012 Rabkin E et al 2012 The ProteOn HTG sensor chip novel surface for stable capture of histidine tagged proteins for protein protein interaction analysis Bio Rad Bulletin 6132 The histidine tag is one of the most
104. gle shift as SPR response workflow enabling the completion of high quality SPR units RU for accurate kinetics experiments with high efficiency The advantages position the ProteOn XPR36 system as an optimal SPR platform in label free drug discovery and structural biology applications with high quality results and excellent cost effectiveness Analyzes up to 36 different protein interactions in a single run on a single chip Measures a variety of experimental conditions simultaneously using parallel flow fluidics Employs One shot Kinetics technology which enables a complete kinetic analysis in a single run This book describes how to apply the ProteOn XPR36 system for SPR analysis of biomolecular interactions It includes technical introductions user guides and tips and techniques ProteOn XPR36 Technology ProteOn XPR36 Experimental Design and Application Guide 1 1 ProteOn XPR36 Technology Overview Surface plasmon resonance SPR is an optical phenomenon that occurs when p polarized light at a certain wavelength and angle is reflected off a thin metal film the gold film coated on a sensor chip under the condition of total internal reflection TIR The light excites surface plasmons in the metal at a certain incident angle The TIR field generates an evanescent wave in the thin metal film that extends hundreds of nanometers from the surface into the medium above in this case the molecules in contac
105. grams Unlike traditional serial flow SPR systems the ProteOn XPR36 system has a parallel flow design that allows for synchronized analyte injection across different flow channels Therefore injection alignment is typically achieved with very good accuracy RE One Shot Kinetics Experiment ProteOn Manager File Edit View Process Analysis Tools Help Nhe 22 Channel Reference gt by H We D B x Double Reference gt r Navigator etics Experiment Instrume AS Muto Process Protoco 4 Injection Alignment gt Run H Baseline Alignment gt Data JL Artifact Removal b amp Manual Alignment Ee Panel Type Remove Processing RE One Shot Kinetics Experiment ProteOn Manager File Edit Dea 2A Tools Help 1 H BO 0 etics Experiment view Process Analysis Channel Reference gt adl Double Reference gt Instrumet AN Auto Process Protoco y gt Run Data JL f Manual Alignment Injection Alignment gt Baseline Alignment gt Auto Artifact Removal b Selected Remove Processing Panel Type Fig 4 5 The automatic injection alignment and baseline alignment functions offered in ProteOn Manager software Baseline alignment adjusts all the sensorgrams to the same zero baseline level along the y axis thus removing slight baseline level differences among the sensorgrams resulting from previous steps Although the automatic baseline al
106. group contains six injections by default Note Blank injections that are used for double referencing must be made from the running buffer with the cosolvent 8 Place the six DMSO dilutions into the instrument at the positions shown in the sample layout Processing and Applying EV Correction Data When processing SPR data collected using a buffer that includes high refractive index cosolvents the data s bulk reference primary reference must first be corrected for excluded volume effects The data should be processed as follows 1 Use the controls in the Data screen to select and group the analyte data for processing 2 Select Channel Reference and choose EVC Calibration Figure 4 27 Interspot Double reference Column Fugu Reference EWC Ev calibration Fig 4 27 Opening the EVC calibration wizard 3 A wizard opens at the bottom of the Data screen Select a row column or interspot reference If you are using a Column or Row reference use the associated dropdown menu to identify which channel the reference data are in 4 In the step list select a minimum of three EVC injections if they are not already selected Figure 4 28 The wizard displays EVC calibration data as thumbnail plots that show a best fit line These plots are accompanied by a table that lists the R values for the best fit lines Choose reference Select EVC injections R value
107. hange in the amount of complex formed over time is linearly related to Ka Ky and the analyte concentration A The complex formation can be further described in terms of response units where the change in response units over time Is again linearly related to k k and A Thus Equation 2 describes the level of response at equilibrium and also the time taken to reach a certain response level during the association phase Dissociation Phase In the dissociation phase the analyte concentration in the flow is suddenly reduced to zero by the injection of running buffer The rate of complex dissociation follows simple exponential decay or first order kinetics Equation 3 is derived in a manner similar to Equation 2 It describes the time taken to reach a certain response level during the dissociation phase as outlined in Figure 4 14 d AB at k A B k AB lt A 0 d AB dt k AB dR dt k R R R e Ro is the signal level at the beginning of dissociation t Fig 4 14 The derivation of Equation 3 ProteOn Manager software offers two options for the Langmuir model simultaneous k kK or off rate analysis The first option is the default choice which fits both the association and dissociation phases for the full set of constants k k and K whereas the second option analyzes only the dissociation phase for k Langmuir with Drift or Mass Transport There are two other kinetic interac
108. hannel for real time double reference Jif ff ff ff gt ERR lt JTPT_py_T I Fig 6 6 The unique interspot referencing A and real time double referencing B in the ProteOn XPR36 system Saving interaction spots and providing high data quality 107 ProteOn XPR36 Experimental Design and Application Guide What are the advantages of using a capture surface for ligand immobilization compared to direct amine coupling of the ligand The captured ligand has a better recovery yield because the capture mechanism results in the correct orientation of the ligand molecules It is also easy to remove the ligand and generate a new ligand surface Capture surfaces can be used for capturing ligands from crude samples where amine coupling surfaces should not Sometimes capture surfaces exhibit drift or leaching of the captured ligands from the chip surface This can be completely resolved using the unique real time double referencing in the ProteOn XPR36 system How do I correct the baseline drift when using a capture surface A capture surface uses a reagent to reversibly capture a ligand to the surface instead of covalently immobilizing the ligand It has two main advantages ease of surface regeneration and compatibility with non purified ligand samples Sometimes capture surfaces exhibit drift or leaching of the captured ligands from the chip surface This can be completely resolved using the unique real time double referenc
109. he degree of success in ligand immobilization can be visualized by observing the sensorgram during the procedure see Figure 5 1 Typical Ligand Buffer Conditions The typical ligand buffer conditions are listed below 1 The pH should be one unit below the pl value 2 The ligand buffer ionic strength should be low and the ligand concentration should be above 0 1 ug ml 3 If a disulfide bond reduction reagent is needed in the ligand buffer TCEP is preferred over DTT because TCEP is compatible with the amine coupling protocol gt 20 000 Deactivation 15 000 10 000 Activation 5 000 Response RU Immobilization O a eee 200 O 500 1000 1 500 Time sec 25 000 a Deactivation 19 400 13 800 Activation Immobilization 8 200 2 600 3 000 200 180 560 940 1 320 1 700 Time sec Fig 5 1 Sensorgram examples of failed A and successful B ligand immobilization by amine coupling Activation Quality Verify the activation quality by 1 Using fresh activation reagents 2 Immobilizing another protein that was previously used under the same conditions with success Ligand Activity If a sufficiently high Rmax is predicted based on the R value but the binding response is much lower than the expected response this indicates low ligand activity on the surface Because the amine coupling method randomly links any fr
110. he running buffer injection time in the dissociation phase is long enough to show adequate response decrease to resolve the dissociation rate constant The choice of appropriate injection conditions including interaction time analyte concentration and injection flow rate is based on the user s understanding of the interaction For example the user can determine the binding affinity and ligand analyte complex stability by obtaining this information from either preliminary experimental trials or literature values This consideration is essential for accurate sensorgram fitting Referencing options should be selected in the experiment design phase as the reference surfaces are created in the ligand immobilization and analyte injection steps A combination of blank surface and blank buffer referencing is uSually applied to yield high quality SPR results This combination is implemented by sequentially subtracting one blank surface reference and one blank buffer reference The ProteOn XPR36 system offers the flexibility of selecting any combination of the four references to optimize data processing Note The steps of data processing and data analysis using ProteOn Manager software are outlined in section 4 7 12 Experimental Design 4 5 Guide to SPR Data Analysis on the ProteOn XPR36 System 4 5 1 Kinetic Analysis To determine the kinetic constants of a biomolecular interaction through SPR analysis the sensorgram must be fitt
111. he same solution at different time points 0 72 hr resolving the fast k AB 1 42 monomers and slow k AB 1 42 oligomers Experimental data binding species 1 binding species 2 binding species 1 fast Ks binding species 2 slow k RU response units Applications Balducci C et al 2010 Synthetic amyloid beta oligomers impair long investigated In order to characterize the binding site on term memory independently of cellular prion protein Proc Natl Acad Sci C C 23 230 USA 107 2295 2300 Mi the PrP fragments i i l 7 panne and Biasini E 2013 Using SPR to characterize the interaction between terminus were captured ON Ihe Chip SUMaCe using the cellular prion protein and AB oligomers Bio Rad ProteOn Webinar an anti Myc antibody AB monomer and AB oligomers Series were injected in the parallel flow channels for interaction Fluharty BR et al 2013 An N terminal fragment of the prion protein analysis Figure 3 18 Further characterization showed binds to amyloid B oligomers and inhibits their neurotoxicity in vivo J Biol Chem 288 7857 7866 the interaction between the N1 terminus and transient AB assemblies It was discovered that the N1 terminus These articles Balducci et al 2010 and Fluharty et al of PrP was necessary and sufficient to facilitate 2013 and the webinar Biasini 2013 describe research the binding to AB oligomers and could block some on a pathway related to Alzeheime
112. his is a completely novel system then choose a very large range of concentrations for an initial scouting experiment This allows you to home in on the concentration range gradually decreasing the concentration range to span 10x above and below the K 4 3 4 Analyte Preparation Knowing your analyte concentration is key as it directly affects k and k Analyte samples should be created using serial dilution into running buffer to minimize bulk effects Take care to avoid vortexing as this will cause bubbles and destroy proteins Samples may be centrifuged for about 15 seconds to ensure that all of the solution is at the bottom of the tubes prior to loading into the instrument 4 3 5 Analyte Injection Parameters Typically analyte injections are performed at a high flow rate of about 50 100 l min this helps to reduce any mass transport effects that may be present if a lower flow rate is used Injections should be performed once the baseline is stable A stabilization steo may be needed in some cases In order to calculate correct binding constants the amount of time alloted to the association phase should be enough to observe a curvature of the binding response and the dissociation phase should be long enough to observe decay in the response Figure 4 3 Concentration range and injection length are long enough to see curvature Concentrations are too low or injection length is too short so only the line
113. ics 57 6 2 Sensorgram 104 i 6 3 Sensor Chips 104 Chapter 4 Experimental Design 59 P 6 4 Experimental Design 106 4 1 Introduction to SPR Experimental Design 60 41 1 ProteOn XPR36 System 60 6 5 Experimental Tips 108 4 1 2 Senet of eee PORCA Are 60 Chapter 7 Quick Guides 111 ta n a 62 7 4 Writing a ProteOn XPR36 Experiment alae y Protocol 112 4 2 1 Conditioning 62 i l 4 2 2 Activation 62 7 2 Running an Experiment 4 2 3 Immobilization 63 with the ProteOn XPR36 System 114 4 2 4 Deactivation 64 7 2 1 Instrument Preparation 114 4 2 5 Stabilization 64 7 2 2 Running an Experiment 114 4 2 6 Ligand Capture Using Capture Proteins 7 2 3 Instrument Maintenance 114 Antibody Screening 64 7 2 4 Import Export Experiment Files 115 ProteOn XPR36 Experimental Design and Application Guide Introduction Explore the World of Parallel Surface Plasmon Resonance Analysis ProteOn XPR36 Experimental Design and Application Guide Measuring Biomolecular Interactions with SPR Surface plasmon resonance or SPR is a biosensor The binding of the analyte to the ligand is tracked technology enabling label free and real time measurement in real time by following the change in SPR signal of biomolecular interactions A typical SPR experiment over time and this time traced graph is called a involves first immobilizing a ligand a biomolecule suchas sensorgram Fitting the sensorgram to a suitable a protein or a nucleic acid to the functionalized s
114. ies such as liposomes and lipoparticles allows the study of native membrane proteins as well as the role of the lipid bilayer of these assemblies in the activity of the membrane proteins Analyzing lipid assemblies also provides insights into lipid protein or lipid small molecule interactions answering critical questions in the fields of drug delivery virology and signal transduction Modified GLC Sensor Chip Lipophilic Surface Chemistry The GLC sensor chip can be modified for capturing lipid assemblies The surface lipophilicity of the chip is adjusted through the amine coupling of an alkyl chain for capturing lipid substances This capture approach provides the flexibility to control the lipophilicity of the chip surface for customized lipid based applications 24 ProteOn Sensor Chips The modified GLC sensor chip provides a traditional lipophilic Surface chemistry for capturing lipid assemblies which allows for typical lipid based applications Bio Rad offers the GLC lipid kit as an all in one kit composed of the GLC sensor chip and the lipid modification kit The advantages of this application kit are summarized below Lipophilic surface chemistry Flexibility in adjusting surface properties Good regeneration capability Low cost High throughput LCP Sensor Chip Hydrophilic Surface Chemistry The LCP sensor chip provides a surface functionalized with NeutrAvidin in a planar configuration that is form
115. ignment function processes entire sensorgrams by default it is possible to align the sensorgrams based on the values in a selected region To define the selected region click and drag in the sensorgram graph Click Process select Baseline Alignment and choose Selected in the submenu to perform baseline alignment for the selected region In addition to the alignment options described above manual correction may be applied to fine tune the sensorgrams To perform the manual sensorgram alignment click Process and select Manual Alignment Individual sensorgrams can then be moved when selected with the mouse Remove the Artifacts Sensorgram artifacts usually spikes caused by tiny air bubbles in the analyte solution are sometimes present and should be removed Note that artifact here refers to a response deviation over a very small time period If a significant portion of the sensorgram deviates from the expected response the trial should be rerun ProteOn Manager software offers automatic artifact removal that flattens these artifacts to restore sensorgram integrity as illustrated in Figure 4 6 Although the automatic artifact removal processes entire sensorgrams by default it is possible to process a selected region of the sensorgrams To define the selected region click and drag in the sensorgram graph Click Process select Artifact Removal and choose Selected in the submenu to perform artifact removal within the selected region
116. in Kinetic screening protein characterization protein quantitation assay optimization and other novel research fields ProteOn XPR36 Experimental Design and Application Guide 3 1 Overview Bio Rad s ProteOn XPR36 protein interaction array system uses surface plasmon resonance SPR technology to detect and monitor biomolecular interactions in real time for label free interaction analysis Binding events are detected by monitoring the change in the SPR signal which is proportional to changes in mass at the sensor chip surface over time as an analyte flows through a microfluidic channel and interacts with a target immobilized to the sensor chip The ProteOn XPR36 system can be used to monitor many different biomolecular interactions including those between antibodies and antigens enzymes and their substrates inhibitors small molecules and drug discovery targets and whole cells and lipid membranes SPR technology is flexible the applications for SPR are vast and experimental design can be tailored to individual needs An additional advantage of the ProteOn XPR36 instrument is that unlike some traditional SPR biosensors the unique XPR technology and 6 x 6 interaction array allow for the simultaneous measurement of up to 36 biomolecular interactions Figure 3 1 XPR technology greatly speeds time to results for traditional kinetic measurements by enabling the patented One shot Kinetics approach whereby up to six targe
117. in a twofold dilution series ranging from 10 0 63 nM RU response units 25 ProteOn XPR36 Experimental Design and Application Guide 2 4 Guidelines for Choosing the Right ProteOn Sensor Chip Which sensor chip you choose depends on a number of experimental parameters To helo decide you may consider the following questions What is the application What kind of information do want to get from my ProteOn experiment What type of samples am working with Proteins Peptides Biotinylated or histidine tagged proteins Small molecules Whole cells DNA Do know anything about the affinity tightness of binding What are the molecular weights in my sample For routine examination of protein protein interactions such as antibody and antigen the GLC chip is a good first choice The GLM or GLH chip can be used as needed if troubleshooting your interaction with the GLC chip suggests that you need a higher capacity surface Knowing the molecular weight of your analyte is also crucial for choosing the right sensor chip as low molecular weight analytes such as small molecules need a higher capacity ligand surface and will benefit from the enhanced sensitivity afforded by the GLH chip An additional consideration to take into account when choosing a sensor chip is whether or not your interaction is mass transport limited in other words whether or not your interaction has a fast associat
118. inding stability compared to that of the traditional NTA surface Figure 6 2 Bio Rad offers two ProteOn sensor chips for various histidine tagged protein applications HTG for compact density large molecule applications and HTE for high density small molecule applications Both the HTG and HTE sensor chips allow easy surface regeneration chip reuse and capture of histidine tagged proteins directly from crude samples a NTA eC P Qp l NTA i C NTA 9 5 ae a a o oO lt a N E E is DS ae KI N NTA Ne O 7 o E a a O O l o A i s Q Ve 7 XN ee os k x er NTA Structure Tris NTA Structure Fig 6 2 Structures of NTA and tris NTA bound to SPR sensor chips The challenge in working with membrane proteins is finding methods to capture the membrane proteins while keeping them active Is there any information about the study of membrane proteins with the ProteOn XPR36 system If the soluble form of membrane proteins is available for use you may immobilize the membrane proteins with the same methods as for other protein targets such as amine coupling and antibody capture However many membrane proteins require a lipophilic environment to maintain the ability to react with biomolecules A common method of maintaining lipophilic environments is to embed proteins in lipid assemblies such as liposomes Biomolecular interactions involving lipid assemblies such as liposomes is an interesti
119. ing level LCP Sensor Chip and Liposome Capturing Kit Refer to the product insert part number 10024332 of the liposome capturing kit for details on how to use this kit Activation Conditioning and Liposome Capture Inject the biotin ssDNA solution for surface activation Then precondition the chip surface with the lipid modification conditioning solution 20 mM CHAPS before liposome capture Next inject the chol dsDNA 1 tagged liposome solution If an additional liposome layer is needed inject the chol dsDNA 2 solution 0 4 uM and allow the signal to stabilize for 5 min then inject the chol dsDNA 1 tagged liposome solution Repeat this step to form multiple liposome layers Table 5 3 LCP chip activation conditioning and liposome capture parameters Volume Flow Rate Injection Reagent Orientation ul ul min 1 3 uM 1 o ENA Vertical 50 30 2 20 mM CHAPS Vertical 150 30 1 mg ml 3 chol dsDNA 1 Vertical 150 30 tagged liposomes 0 4 uM 4 chol dsDNA 2 Vertical 50 30 1 mg ml 5 chol dsDNA 1 Vertical 150 30 tagged liposomes Note Injections 4 and 5 are optional GLC Lipid Kit Refer to the product insert part number 10023826 of the GLC lipid kit for details on how to use this kit Surface Modification Use the activation and deactivation reagents from the ProteOn amine coupling kit Inject 1x EDC sulfo NHS lipid modification solution and 1 M ethanolamine HCI sequentially Table 5 4 GLC surface modificati
120. ing in the ProteOn XPR36 system The solution to baseline drift is referencing the sensorgram to a blank running buffer injection Figure 6 7 The ProteOn XPR36 system features unique real time double referencing to correct this effect providing the best referencing accuracy The ProteOn XPR3 amp 6 system is the only SPR biosensor to feature real time double referencing that runs simultaneously with ligand analyte interactions Analyte QO Q Q y Interaction Ligand Capture reagent i ee a i J Reference Ligand is washed off in dissociation causing l Baseline arit Corrected Interaction Fig 6 7 The baseline drift when using a capture surface can be completely resolved using the real time double referencing option in the ProteOn XPR36 system For robust kinetics how many concentration points should be analyzed Kinetic analysis requires at least three dose responsive sensorgrams A single analyte injection on the ProteOn XPR36 system collects a set of six sensorgrams five sensorgrams if the real time double referencing is applied that is one per analyte concentration In the data analysis section of the software the user may select the three to five best sensorgrams for analysis using the following criteria Good sensorgram reproducibility An analyte concentration in the range of 0 1K 10K 6 5 Experimental Tips Should I degas the buffers and samples It is not required to degas the running buffer becau
121. ing the ProteOn XPR amp 6 system aliquots of a solution of AB 1 42 monomer were taken at different time points and injected to the system to analyze the interaction with 4G8 antibody As time passed the aliquot contained varying concentrations of synthetic AB 1 42 monomer and spontaneously formed AB 1 42 oligomers and AB 1 42 fibrils The interaction sensorgrams were deconvoluted to obtain the concentration ratio between AB 1 42 monomer and AB 1 42 oligomers that was verified by combining SPR with chromatography Figure 3 17 It was shown that the immunoassay could be used to analyze the formation of AB oligomers thus allowing the evaluation of inhibitors and effects of AB mutations as well as the potential detection of native AB oligomers in biological samples 4G8 antibody immobilized AB 1 42 aliquots Fast Koi 6 3 x 10 s7 Slow Ks 1 1 x 1074 s7 A t 0 B t 2hr 600 600 D T 400 T g g 5 200 5 Q Q a a 04 T T T O 300 600 O 300 600 Time sec Time sec C t 5hr D t 8hr 600 5 D gt o aa oO Oo dp dp Cc Cc O O Q Q N N oO oO cm O 300 600 O 300 600 Time sec Time sec E t 24hr F t 72 hr 600 600 D gt am 400 4 am 400 oO oO dp dp 5 200 4 5 200 4 Q Q icp icp oO oO ao ao O Of O 300 600 O 300 600 Time sec Time sec Fig 3 17 Binding of AB 1 42 aliquots taken from t
122. injection B ii for the analyte injected in the reference channel showing NSB the analyte response exhibits curvature during the injection and does not return to zero at the end of the injection RU response units Q l Analyte y Electrostatic NSB Ligand analyte interaction Electrostatic NSB Ligand analyte interaction a E gt Analyte Ligand Ligand b d Surface layer Surface layer Gold substrate Surface layer Fig 5 4 Electrostatic NSB on the chip surface 97 ProteOn XPR36 Experimental Design and Application Guide Non Electrostatic NSB Sources of NSB also include chemical interactions of the analyte or other components with the binding layer such as hydrophobic interactions hydrogen bonding or binding to nanoscopic areas of exposed gold on the surface Figure 5 5 This type of NSB termed non electrostatic NSB is usually observed when sticky or crude analyte samples are applied When using the HTG or HTE chips non electrostatic NSB can also be caused by proteins with a sequence containing a few adjacent histidine residues which have a low affinity for the nickel ll activated tris NTA surface Molecules that can potentially exhibit electrostatic and non electrostatic NSB are listed in Table 5 6 Analyte Non electrostatic NSB Ligand analyte Non electrostatic Ligand analyte interaction NSB interaction Ligand b d b d gt Ligand b d a D Surface
123. ion rate High Capacity surfaces exacerbate a mass transport limited interaction because high density of ligand on the sensor chip depletes the analyte in the surrounding solution very quickly whereas compact density of ligand mitigates the influence of mass transport by decreasing the rate at which analyte is depleted from the surrounding solution Thus the GLC chip with a compact capacity surface may be more suitable than the high capacity GLH chip in dealing with a mass transport limited interaction If the ligand is biotinylated or contains a histidine tag the NLC HTG and HTE chips can be used to immobilize ligand on the chip surface without the need to use amine coupling An additional benefit of using the NLC HTG and HTE chips is that the ligand is immobilized on the chip surface at a specific orientation as opposed to several random orientations with amine coupling 26 ProteOn Sensor Chips 21 ProteOn XPR36 Experimental Design and Application Guide d I ey ney T fa i a Applications CHAPTER 3 The ProteOn XPR36 system monitors many different biomolecular interactions including those between antibodies and antigens enzymes and substrates small molecules and drug discovery targets and whole cells and lipid membranes Ihe applications are vast and flexible and the experimental design can be tailored for individual needs The ProteOn XPR36 system has been widely accepted and used
124. is and other applications Refer to Chapter 2 for details on the surface chemistry of each chip For more information about the ProteOn XPR386 instrument and instructions for running experiments refer to Chapters 1 and 4 The ProteOn family of chips features outstanding performance in kinetic analysis high binding capacities high sensitivity for the detection of low molecular weight analytes uniform spot to spot response minimal baseline drift barcodes and long term storage stability Each ProteOn sensor chip is suitable for particular applications including the following ProteOn GLC sensor chip for protein protein interaction analysis ProteOn GLM sensor chip for protein small molecule and protein protein interaction analysis ProteOn GLH sensor chip for protein small molecule interaction analysis ProteOn NLC sensor chip for DNA protein and protein protein interaction analysis ProteOn HTG sensor chip for protein protein and protein peptide interaction analysis ProteOn HTE sensor chip for protein small molecule interaction analysis ProteOn LCP sensor chip for capturing lipid assemblies for lipid protein lipid small molecule and membrane protein protein interaction analysis Bag Qa The sensor chip cartridge label contains the following information Expiration date Chip type Barcode Catalog number Storing Sensor Chips Store chips a
125. istry for a particular application ProteOn liposome capturing kit This kit includes a new LCP sensor chip that is designed for use with the ProteOn LCP capturing reagent kit Figure 6 4 This kit provides a novel hydrophilic surface chemistry that allows for advanced applications such as minimizing lioophilicity based nonspecific binding and capturing liposomes that are difficult to analyze with the traditional approach It is possible to capture multiple layers of lipid assemblies for additional sensitivity 9 ie e cae Kinetic j PoS ES i analysis LCP chip LCP chip Hybridization of the Biotin ssDNA two DNA strands Biotin ssDNA Chol dsDNA 1 liposome Fig 6 4 Workflow for liposome capture using the ProteOn liposome capturing kit based on a novel hydrophilic surface chemistry The LCP chip surface is saturated with single stranded biotinylated DNA molecules and liposomes tagged with cholesterol labeled double stranded DNA molecules are captured to the surface through DNA hybridization For the details of reagents and techniques in this graph refer to Bio Rad bulletin 6161 6 4 Experimental Design How do design an SPR experiment and what factors should be taken into consideration There are two major steps in an SPR experiment ligand immobilization and analyte injection All the factors affecting these two major steps including the pre steps and post steps to enhance the performance of the
126. ity of tris NTA B complex is distinguished in the two different sensor chips HTG for compact density and HTE for high density Figure 2 B and C The HTG and HTE sensor chips allow easy surface regeneration chip reuse and capture of histidine tagged proteins directly from crude cell lysates The HTG sensor chip is an ideal choice for protein protein and orotein peptide interaction analysis and the HTE sensor chip for protein small molecule interaction analysis Figure 2 8 Bio Rad offers the HTG and HTE reagent kit for use Time min with the HTG and HTE sensor chips Analyte Prism Response RU Fig 2 6 ProteOn NLC sensor chip A the NeutrAvidin modified alginate coating on the NLC sensor chip B sensorgrams of the interaction between an antibody Fab fragment and biotinylated MHC Tyr antigen using the NLC sensor chip MHC I was captured to approximately 800 RU and the Fab was injected in a twofold dilution series ranging from 500 31 nM RU response units 23 ProteOn XPR36 Experimental Design and Application Guide A Ta n AMS 7 en _ Og gaa D a amp O 7 NTA N 1 a gt Q gt a NTA 5 ee U l O o i A gt yas J M uN ERI EIA X 2 O h l bon a o aE a e o J se Ve A N 2 ae A B Analyte LP ALP LP LP LP vert Go Voo Incident light Detector C Analyte 2D D gt tris NTA O Detector Incid
127. k File and select Open to open the database browser Select an experiment and press Open to display the z E E sensorgrams In the Navigator sidebar select the Data tab to display the sensorgrams The software may automatically complete x i Perens BES Open file this step te Errea Note If the analysis is performed immediately following the 2h EES R experiment start from the next step C x pansies Open the Panel Type screen and select the step type chosen for sin z analysis Click Apply and then Close ae PlAneite z Cj tirk gt Cir C oe Q 2 Open the Protocol Step screen and select the desired step to ear Protocol step a display Click Apply and then Close 3 Elone shot netics Borman Open the Data Grouping screen and select the appropriate Data grouping 2 ping PESE Miro Groupieg grouping option Press Apply and then Close Ooy by Anat amher 2 Oca by Suse hesba Groupeng h 00 Not Combine Q O q C Com A In the menu bar click Process and select Auto Process to 5 Auto process automatically process the sensorgrams This processing includes oro we EI antes a S A artifact removal injection alignment and baseline alignment ete manens pane s eee yr fm E Qeorie Migrert Oo z 2 2 In the menu bar click Process select Channel Reference and x 1st reference choose the appropriate blank surface reference namely Channel pa Oonan 3 a D Reference in the submenu Pang
128. ked questions on how to use the ProteOn XPR36 system ProteOn XPR36 Experimental Design and Application Guide 6 1 Basics What is SPR What does SPR measure SPR is surface plasmon resonance It senses the refractive index change mass change within a thin layer on the surface of a metal that is in contact with a dielectric medium What applications can the ProteOn XPR36 system be used for Real time kinetic analysis of biomolecular interactions Equilibrium analysis for affinity constant determination Protein concentration quantitation What do the SPR terms sensor chip surface ligand analyte and capture reagent mean Sensor chip surface or surface a metal surface coated with a polymer where SPR is measured The surface is located on an SPR sensor chip Ligand an interaction reagent immobilized on the surface also often referred to as the target Analyte an interaction reagent flowed over the ligand immobilized to the surface Capture reagent areagent immobilized to the surface that is used to capture the ligand by biological interactions It is used to reversibly immobilize the ligand to the surface 6 2 Sensorgram What are the descriptors used to define specific regions of an SPR sensorgram Which regions are used to calculate the kinetic and equilibrium constants k Ky and K The association phase shows second order kinetics and the dissociation phase shows first order kinetics Th
129. l 2006 Exploring One shot Kinetics and small molecule analysis using the ProteOn XPR36 array biosensor Anal Biochem 358 281 288 Bravman T et al 2008 The ProteOn XPR36 array system high throughput kinetic binding analysis of biomolecular interactions Cell Mol Bioeng 1 216 228 Turner B et al 2008 Applications of the ProteOn GLH sensor chip Interactions between proteins and small molecules Bio Rad Bulletin 5679 These articles Bravman et al 2006 Bravman et al 2008 and Turner et al 2008 describe how the ProteOn XPR36 system was used to investigate the interaction between carbonic anhydrase isozyme CAII and a series of Known small molecule sulfonamide inhibitors Kinetic screening results were obtained quickly using the One shot Kinetics approach Figure 3 10 The measured affinity of nine inhibitors were consistent with those determined using isothermal titration calorimetry Table 3 1 Temperature dependence of the kinetics was also measured and Van t Hoff plot was obtained to determine the thermodynamics of the CAll carboxybenzenesulfonamide CBS interaction In addition the binding of a 95 Da methylsulfonamide inhibitor to the ligand CAII was measured showing the high sensitivity of the ProteOn XPR36 system for small molecule interaction analysis 35 ProteOn XPR36 Experimental Design and Application Guide
130. l 30 ul min 5 50 mM NaOH Vertical 30 ul 30 ul min 6 100 mM HCl Vertical 30 ul 30 ul min NLC Chip Step Reagent Orientation Volume Flow Rate 1 50 mM NaOH Horizontal 30 ul 30 l min 2 1M NaCl Horizontal 30 ul 30 ul min 3 50 mM NaOH Vertical 30 ul 30 l min 4 1 M NaCl Vertical 30 ul 30 ul min HTG and HTE Chips Step Reagent Orientation Volume Flow Rate 1 0 5 SDS Horizontal 30 ul 30 ul min 2 50 mM NaOH Horizontal 30 ul 30 l min 3 100 mM HCI Horizontal 30 ul 30 ul min 4 300 mM EDTA Horizontal 100 ul 30 ul min 5 0 5 SDS Vertical 30 ul 30 ul min 6 50 mM NaOH Vertical 30 ul 30 ul min 7 100 mM HCl Vertical 30 ul 30 ul min 8 300 mM EDTA Vertical 100 ul 30 ul min LCP Chip Used in ProteOn Liposome Capturing Kit Step Reagent Orientation Volume Flow Rate 1 1 3 uM biotin ssDNA Vertical 50 ul 30 ul min 2 20 mM CHAPS Vertical 150 ul 30 ul min Step Type Regenerate Orientation Refer to the tables 3 Immobilization This phase immobilizes a ligand to the chip surface through either direct covalent attachment or binding to a capture reagent Consider the following factors before immobilizing ligands Desired ligand immobilization level determine the immobilization level R or amount of ligand immobilized on the chip surface using the following equation Ma Birac aa R L Rmax IS the theoretical maximum response when the ligand interacts with an analyte Ma is the molecular weight of the analyte M is th
131. le The technical note Bronner et al 2011 provides an This webinar Willis 2009 provides an overview of the introduction to the novel liposome immobilization Integral Molecular lipoparticle technology Lipoparticles technology called the MemLAYER technology are virus like particles that contain high levels of the Immobilization protocols for liposomes and lipoparticles protein of interest The protein is correctly folded using this technology in the ProteOn XPR36 system homogenous stable and highly active This offers a were shown The method of membrane protein antibody unique solution for those researchers looking to work interaction analysis is investigated by comparing other with membrane proteins Combining this technology lipoparticle immobilization approaches The screening with the ProteOn XPRS6 system offers an easy to use of drug candidates against malaria proteins using the robust approach to screen antibody therapeutics against novel SPR based approach is discussed MemLAYER membrane proteins in a high throughput manner technology is employed in the ProteOn liposome capturing kit Figure 3 30 50 Applications A Amine Coupling D MemLAYER Capture Single Layer 100 250 Nip Da A ka 80 J wr ye ee Sr has 200 f IMANAN POOP 3 60 5 150 cc JL NT PRP Toe a oO 0 AH 100 oj wee ny AN a 7 a 20 50 0 phy i 0 20 l 50 2
132. lected Protocol Experiment box D Select the chip initialization method and press Initialize Chip For glycerol initialization follow the Click Run to start the experiment pop up instruction to place the normalization solution Note If there is any non timeout pause step in the protocol you will in the instrument have to click Run in that step to continue the experiment E Wait until the Initialization Status box shows Chip 7 2 3 Instrument Maintenance Initialized Type indicating that the chip initialization is 1 Run post experiment maintenance Skip this step if the completed sensor chip will be reused immediately A In the Navigator panel go to the Instrument tab and 7 2 2 Running an Experiment select Instrument Control Click Eject to eject the 1 Write a protocol sensor chip Note Refer to section 7 1 B Insert an MNT chip Wait until the Initialization Status box shows Maintenance Chip The software will automatically choose the Maintenance screen A In the menu bar click File and select New to open the database browser Select New Protocol and press the New Protocol button to start with a blank protocol or select an existing protocol and press New Protocol to copy the selected protocol C Click Post Experiment Follow the pop up instruction to load the reagents and click Next to Note In the database browser Protocol means the set of instrument start the maintenance protocol parameters sample information
133. lies Modified GLC and LCP 24 4 5 2 Equilibrium Analysis 76 2 4 Guidelines for Choosing the Right 4 5 3 Concentration Analysis m7 ProteOn Sensor Chip 26 4 5 4 Report Point 1T 4 5 5 Data Presentation 78 Chapter 3 Applications 29 4 5 6 Sensorgram Appearance 79 31 Overview 30 4 5 7 Quality Standards for SPR Results 80 4 6 How to Perform Excluded Volume w 1 ee Screening if Correction on the ProteOn XPR36 3 2 2 Epitope Binding and Mapping 33 Protein erection System 81 3 2 3 Quantikinetics 34 4 7 Data Processing and Analysis Flowchart 84 3 2 4 Drug Compound Screening 35 4 8 Options for Dataset Export 86 3 3 Biomolecule Characterization 40 i A 3 3 1 Structural Biology 40 Chapter 9 Tips and Techniques 89 3 3 2 Thermodynamics and Energetics 46 5 1 Tips for Using ProteOn Sensor Chips 90 3 3 3 Histidine Tagged Protein Analysis 47 l l l 3 3 4 Nucleic Acid Interaction Analysis 48 5 2 Running Experiments with Sensor Chips 91 3 3 5 Lipid Membrane and Membrane Protein Analysis 49 5 2 1 Conditioning 91 3 3 6 Cell Antibody Interaction Analysis 52 5 2 2 Ligand Immobilization 92 3 3 7 Regenerable Biotin Capture Surface 52 5 2 3 Troubleshooting Ligand Immobilization 94 3 3 8 SPR MS Analysis 53 5 2 4 Stabilization 96 3 4 Biological Assays 54 ps slat lila ei 3 4 1 Assay Design and Optimization 54 g eae Biosimiar Assessmeni 56 Chapter 6 Frequently Asked Questions 103 3 5 Biomedical Applications 56 6 1 Basics 104 3 5 1 Vaccine Characterization 56 3 5 2 Clinical Diagnost
134. ligand and or chip surface Regeneration conditions should be optimized for each interaction The reproducibility of repeated analyte injections is typically used to check the performance of regeneration With a good regeneration protocol the sensorgrams of repeated analyte injections should overlap when viewed in the same window GLX and NLC Sensor Chips For GLX and NLC chips the ligand is bound to the chip surface by covalent or very high affinity noncovalent linking Regeneration is used to remove the analyte while keeping the ligand active on the chip surface The regeneration conditions should be optimized to a balance that is strong enough to completely remove the analyte but not so harsh as to damage the ligand Some recommended conditions for different interaction systems are listed below Table 5 8 GLX and NLC chip regeneration reagents Ligand Analyte Recommended Reagent 10 mM glycine pH 1 5 3 0 1 phosphoric acid Peptide 0 01 0 5 SDS nucleic acid Proteinpeptide 5 10 mM NaOH 5 10 mM NaOH deionized water Protein antibody Protein peptide Nucleic acid Nucleic acid HTX Sensor Chips The captured ligand can be stripped off and replaced with fresh ligand by a highly efficient regeneration step using 300 mM EDTA pH 8 5 Once the ligand is removed the chip can be reactivated to capture new ligands Table 5 9 HTX chip regeneration conditions Flow Rate Injection Reagent Orientation Vo
135. lize 36 different ligands in a stepwise immobilization procedure designed for the high throughput epitope mapping and binding of antibody antigen interactions Abdiche et al 2011 The 6 x 6 interaction array of the ProteOn sensor chips also allows for inline referencing whereby data from unmodified spots in between the immobilized ligand spots on the sensor chip are used to subtract out artifacts such as noise and baseline drift This inline referencing is superior to referencing with a separate flow cell and means the ProteOn XPR36 system can collect high quality SPR data at the low signal to noise ratios often seen with small molecule analytes 1 2 What Kind of Information is Obtained with the ProteOn XPR36 system As an SPR biosensor platform the ProteOn XPR386 system provides a wide variety of important information on biomolecular interactions such as the specificity affinity qualitative ranking kinetics and thermodynamics of binding The ProteOn XPR36 system can be used in pharmaceutical drug discovery antibody characterization immunogenicity testing the development and manufacture of biologics or for clinical research It could also be used in other fields where there is a need for label free characterization of biomolecular interactions Key applications include Quantification of binding affinity and kinetics Determination of binding specificity and the number of binding sites Characterization of the m
136. lly a reference protein unrelated to the ligand should be bound at the same density as the ligand 2 Minimizing NSB by optimizing the buffer conditions and surface chemistry A No NSB i Analyte on ligand channel 200 gt 160 a 120 4 Cc O a 80 a 40 O 40 O 40 80 120 160 200 240 Time sec ii Analyte on reference channel 200 gt 1604 a 1207 Cc O Q 80 ir 40 O 40 O 40 80 120 160 200 240 Time sec Electrostatic NSB NSB is most commonly caused by the electrostatic attraction of a positively charged analyte or other sample components to the negatively charged surface layer of the sensor chip Figure 5 4 This type of NSB termed electrostatic NSB is common when the analyte is a protein with a pl higher than the pH of the running buffer B NSB i Analyte on ligand channel 600 D a 400 e O Q D O a 100 O 100 200 300 400 Time sec ii Analyte on reference channel 600 D a Oo 400 ie O O 200 O 100 O 100 200 300 400 Time sec Fig 5 3 Comparison of responses on reference surfaces showing only bulk effect a refractive index difference between the sample buffer and running buffer A or exhibiting NSB B A ii for the analyte injected in the reference channel showing no NSB the analyte response is flat during the injection and returns to zero at the end of the
137. lternative For fine tuned control of the surface modification level it is recommended to vary the undecylamine concentration by diluting the lipid modification solution undecylamine in the lipid modification conditioning solution CHAPS Liposome Capture Before liposome capture condition the chip surface with the lipid modification conditioning solution 20 mM CHAPS and then inject the liposome solution Table 5 5 Liposome capture parameters Volume Flow Rate Injection Reagent Orientation ul ul min 1 20 mM CHAPS Vertical 150 30 2 1 mg ml liposomes Vertical 150 30 When injecting ligand concentrations between 0 5 and 25 ug ml are typically used with a flow rate of 30 ul min and a contact time of 1 14 min These conditions will produce signals of up to 5 000 RU on the HTG chip and 12 000 RU on the HTE chip Please note that when capturing ligands from crude media various other proteins may also be adsorbed to the chip surface mainly due to nonspecific interaction with nickel II ions Fortunately these proteins will not interfere with the kinetics because ligand analyte interactions are specific However the presence of nonspecifically bound lysate proteins will make accurate determination of ligand binding levels difficult given that the observed signal is the sum of the ligand signal and the signal from other bound proteins 5 2 3 Troubleshooting Ligand Immobilization Amine Coupling with GLX Chips T
138. lume ul ul min 300 mM 1 EDTA pH8 5 Vertical 400 30 EDTA may not completely remove nonspecifically adsorbed proteins because they are adsorbed to the surface not only via the nickel II ions but for example also by electrostatic interactions In such cases other ProteOn regeneration solutions such as 50 mM NaOH and 100 mM HCl may be needed along with EDTA to regenerate the surface LCP Chip and Liposome Capturing Kit Regeneration is accomplished by DNA dehybridization using the following conditions Injection 2 is optional because it is used to remove the remaining lipid assemblies if the regeneration is incomplete with injection 1 For the first injection use a freshly prepared 8 M solution of urea in deionized water Table 5 10 LCP chip regeneration conditions Flow Rate Injection Reagent Orientation Volume ul ul min 1 8 M urea Vertical 150 30 20 mM i 2 CHAPS Vertical 150 30 If urea is not available inject deionized water GLC Lipid Kit When using the GLC lipid kit injecting lipid modification solution 20 mM CHAPS will regenerate the chip surface Table 5 11 GLC chip regeneration conditions Flow Rate Injection Reagent Orientation Volume ul ul min 20 mM 1 CHAPS Vertical 150 30 100 Tips and Techniques 101 ProteOn XPR36 Experimental Design and Application Guide 102 Frequently Asked Questions CHAPTER 6 Answers are provided to frequently as
139. mine coupling method is the most typical in SPR experiments The ProteOn amine coupling kit contains all the reagents needed for amine coupling of proteins or peptides to the sensor chip Major Steps for Covalent Immobilization 1 Conditioning of sensor chip 2 Immobilization of ligand activation immobilization and deactivation 3 Stabilization of ligand surface 4 2 1 Conditioning Conditioning is recommended for new sensor chips and can generally improve data quality by cleaning the new chip surface encouraging rapid stabilization of the baseline prior to the start of the experiment Sensor chip conditioning is optional Conditioning is performed following the sensor chip initialization process using ProteOn regeneration solutions refer to Chapter 5 4 2 2 Activation In this step reactive groups are formed on the sensor chip surface The ligand of interest such as a protein is then attracted to the surface and binds through amine coupling Any primary amine within a protein sequence can bind lysine residues and the N terminus To create these reactive groups an activation solution is applied to the surface This activation solution consists of an equivolume mixture of two reagents 1 ethyl 3 3 dimethylaminopropyl carbodiimide EDC and N hydroxysulfosuccinimide Sulfo NHS These two reagents are part of the ProteOn amine coupling kit and are prepared by addition of 7 5 ml water to each reagent bottle to make 400 mM
140. n a single sensor chip This technical note Tabul et al 2010 describes the rapid optimization of immobilization conditions for protein kinase targets 938 and Erk2 to ensure that the immobilized molecules remain in an active state p38 SB203580 p38 D 2 z oO nn 2 g fo Cc O O a Q a as or 20 24 68 112 156 200 Time sec Time sec p38 p38 PD169316 D gt Ta 2 2 S O Q 2 a 2 cc PAT Ep Pry PA 4 f AUREA S W S BY 20 20 34 88 142 196 20 64 148 232 316 400 Time sec Time sec Fig 3 12 Ligand protected immobilization left with the addition of inhibitors SB203580 and PD169316 shows preserved p38 kinase activity compared to direct immobilization right RU response units Erk2 ATP Erk2 80 80 60 60 c 40_ T 40 g D a S 2 A 20 gt AAA LA AS 5 D 0 m Q KpnI X VANAD X PL Aaah Ma ol AN 20 20 20 4 28 52 76 100 20 4 28 52 76 100 Time sec Time sec Fig 3 13 Ligand protected immobilization left with the addition of ATP does not impact Erk2 kinase activity compared to direct immobilization right RU response units 38 Applications Luo R et al 2011 Novel ProteOn sensor chips with high stability and high selectivity for label free polyhistidine tagged protein interaction analysis Poster presented at Antibody Engineering and Therapeutics
141. n of the bound complex Once the complex AB forms it can either dissociate to unbound ligand B and free analyte A or change to the new conformation AB However the complex AB must return to the first complex AB before dissociating into unbound ligand and free analyte The two state conformation model is very useful for describing an allosteric binding effect where binding of analyte to ligand a substrate or inhibitor binding to an enzyme for example results in a conformational change Parameter Setting In ProteOn Manager software there are two parameter setting options for kinetic analysis 1 the choice of Fitted or Constant parameters and 2 the choice of Global Grouped or Local sensorgram fitting scopes as indicated in Figure 4 15 Grouped Grouped v Grouped Fitted Grouped Grouped Fig 4 15 The parameter setting options for kinetic analysis The fitted parameters are variables in the sensorgram fitting whereas constant parameters are fixed at their initial values The three sensorgram fitting scopes global grouped and local are defined and compared in Table 4 3 When setting the parameter fitting types the initial values of all parameters can be changed to start the sensorgram fitting from a closer point to the result This option may be applied in sensorgram fitting with complex models to reduce computational demands Table 4 3 shows the results of an experiment with
142. nd pl to facilitate charge attraction between the positive ligand and the negative chip surface Low ionic strength is also required to enhance the charge attraction Note It is possible to use the co inject function in the ProteOn XPR36 system to remove the manual mixing of the activation reagents Refer to the article Ligand Immobilization in Protein Interaction Studies An Unattended Amine Coupling Protocol with Automatic Coinjection Activation at bioradiations com May 16 2012 Step Details Step Type EDC sulfo NHS for amine coupling chip or NiSO for HTG and HTE chips Activate Ligand Ligand Ethanolamine for amine coupling chip or biotin for NLC and LCP chips Deactivate Orientation Vertical Note NLC and LCP chips do not need activation and deactivation with biotin is optional HTG and HTE chips need activation but not deactivation 4 Stabilization This phase removes unattached ligand from the chip surface and stabilizes the baseline after multichannel module MCM rotation Allowing the baseline to stablize for 30 min is recommended to obtain high quality kinetic analysis The phase includes performing one or more injections which may consist of either or both of the following For NLC chip injection of 1 M NaCl following the running buffer is recommended Y Running buffer Z Any regeneration solution compatible with the ligand Step Details Step Type Running buffer Blank Regenera
143. nding domain was immobilized on an NLC chip and single and polymerized forms of ubiquitins were used as analytes Marchese 2011 Dose response sensorgrams of the interaction of the peptide and the analytes were observed Figure 3 22 200 Response RU 100 100 O 100 200 300 400 Time sec Fig 3 22 Sensorgrams showing dose response analysis of the interaction between a peptide and ubiquitins RU response units High Affinity Proteasome Activator Interaction Analysis Stadtmueller BM et al 2010 Structural models for interactions between the 20S proteasome and its PAN 19S activators J Biol Chem 285 13 17 This article Stadtmueller et al 2010 describes the structural models of the binding between the PAN 19S activators and the 20S proteasomes from T acidophilum and S cerevisiae The models were determined by analyzing the interactions between PA26 variants and each proteasome in a workflow based on label free analysis The ProteOn XPR36 system was employed to characterize the binding kinetics and affinity between the two types of biomolecules The result shows the ability of the ProteOn XPR36 system to detect dissociation constants at the level of 1 x 10 s1 and equilibrium constants at the level of 1 pM which are the typical limits for label free biosensors in analyzing high affinity biomolecular interactions 45 ProteOn XPR36 Experimental Design and Application Guide 3 3 2 Therm
144. nding to histidine tagged Erk2 were screened based on the equilibrium analysis Figure 3 14 Equilibrium analysis ATP o Staurosporin JAK3 inhibitor VI Z Purvalanol B E Amino purvalanol A 2 ES 1 Naphthyl PP1 9 a eee o I I I 30 40 50 60 7O 80 90 100 Concentration UM Inhibitor MW Da Kp UM R naxx RU ATP positive control 507 2 16 8 41 3 Staurosporin 466 5 12 7 156 7 JAKS Inhibitor VI 383 4 10 2 103 6 Purvalanol B 432 9 1 5 56 2 Amino Purvalanol A 403 9 9 5 93 7 1 Naphthyl PP1 317 4 18 7 55 1 With some inhibitors the highest concentration signal was omitted from the analysis since it was higher than the theoretical Rmax This could result from aggregate formation at high analyte concentrations Fig 3 14 Experiment screening small molecule inhibitors against a polyhistidine tagged enzyme The high quality results show the potential of using the HTE chip in drug screening The ligand was captured on the HTE chip surface and the response reached 12 700 RU The capture uniformity and the baseline stability were excellent Fixation of the ligand to the surface is not necessary Analyte concentrations ATP 100 50 25 12 5 and 6 2 UM Staurosporin 50 16 7 5 6 1 9 and 0 6 uM JAKS Inhibitor VI 16 7 5 6 1 9 0 6 and 0 2 uM Purvalanol B 50 16 7 5 6 1 9 and 0 6 uM Amino purvalanol A 50 16 7 5 6 1 9 and 0 6 uM 1 Naphthyl PP1 50 16
145. ng direction in biological research today An essential purpose of using lipid assemblies is attaining native membrane proteins embedded in the lipid bilayer of these assemblies by which the activity of the membrane proteins is maintained In order to facilitate the interaction analysis with lipid assemblies two ProteOn kits have been developed GLC lipid kit and the liposome capturing kit These kits facilitate 105 ProteOn XPR36 Experimental Design and Application Guide O O O O 0909 Liposome Osoppo EDC NHS Alkylamine Ethanolamine CHAPS feo amp 2 SaL ap A Liposomes LaLa 0 amp b o Kineta A w S ba ee bs Y o Y W 0 J Analysis GLC chip Modified GLC chip Modified GLC chip Fig 6 3 Workflow for liposome capture using the ProteOn GLC lipid kit based on the traditional lipophilic surface chemistry The lipophilicity of the GLC chip surface is adjusted through surface modification in order to capture lipid assemblies such as liposomes membrane involved interactions analysis such as lipid protein interactions lipid small molecule interactions and membrane protein protein interactions which are usually considered difficult targets in label free interaction analysis ProteOn GLC lipid kit this kit is based on the traditional approach of capturing liposomes using a modified lipophilic GLC sensor chip surface Figure 6 3 It also provides the flexibility to adjust the surface chem
146. ng events in real time and the accurate measurement of both association k and dissociation k rate constants This additional information can be helpful in designing potent agonists antagonists with a fast k and slow k or for understanding quantitative structure activity relationships of small molecules The unique One shot Kinetics approach of the ProteOn XPR386 system allows for robust kinetic characterization from a single analyte injection In addition biomolecular interaction thermodynamics can be characterized by using the ProteOn XPR36 system to quantify kinetics at different temperatures 3 3 1 Structural Biology The ProteOn XPR36 system has wide applications in structural biology and biophysics to pinpoint structures in small molecules and regions on proteins that are responsible for binding Because SPR obtains information on both the association and dissociation phases of the biomolecular interaction it allows for discovery of quantitative structure activity relationships based on the effects of specific structures SPR benefits include Highly efficient mutagenesis workflow for structural biology Compatible with crude samples Flexible experimental configuration Efficient experimental optimization Published Applications Characterization of the Interaction of TEM1 and BLIP Bronner V et al 2005 Mechanisms of protein protein binding double mutant cycle analysis using the ProteOn XPR36 system Bio R
147. ng of k only Langmuir with drift simple 1 1 biomolecular interaction with a constant baseline drift taken into The 6 x 6 interaction array brings these benefits to SPR experiments account Experimental versatility Langmuir with mass transport limitations simple High productivity 1 1 biomolecular interaction that takes into account High data quality the rate of diffusion of analyte from the bulk to the chip surface Bivalent analyte one analyte has two binding sites to one ligand Heterogeneous analyte two analytes compete for binding to one ligand Heterogeneous ligand one analyte binds to two ligands Two state conformation accounts for a change in conformation of the binding complex that occurs after the analyte binds In addition it is possible to calculate the affinity value K using equilibrium analysis in which the equilibrium responses at different analyte concentrations are filled to a simple saturation binding model a PI ee PIEP IEI REI PI rare MIA 13 ProteOn XPR36 Experimental Design and Application Guide Experimental Versatility Multiple Experimental Configurations and Fast Qualitative and Quantitative Assays Kinetic Characterization 1 to 1 In kinetic characterization experiments the optimization of experimental protocols is usually the most labor intensive
148. njection of the analyte is finished Running buffer flows over the sensor chip surface and washes off the analyte bound to the ligand A convex decreasing response curve is produced Depending on the binding kinetics of the interaction partners and the experimental conditions the decreasing response curve may or may not return to baseline which indicates complete dissociation of the analyte from the ligand surface Baseline Association Dissociation RU Time Fig 4 4 A typical SPR sensorgram displaying the three sensorgram phases RU response units Kinetic analysis requires the presence of all three ohases whereas equilibrium and concentration analyses do not However note that 1 the association phase must reach the interaction equilibrium plateau for an equilibrium analysis and 2 the beginning of the association phase must be linear to reveal mass transport limitations to kinetics in a concentration analysis 4 4 2 Sensorgram Display In the 6 x 6 experimental configuration of the ProteOn XPR36 system 36 sensorgrams are produced simultaneously in each SPR experiment In the sensorgram display by default the sensorgrams are grouped by ligand in six sensorgram windows or graphs In the One shot Kinetics approach each graph contains a set of six dose response sensorgrams for a dilution series of the analyte interacting with six identical ligand surfaces During data processing the grouping of s
149. nning buffer or regeneration solutions removes any noncovalently bound ligand molecules from the chip surface Thus stabilization creates the stable baseline required to perform the interaction analysis Stabilization involves performing one or more injections of either the running buffer or regeneration solutions that do not affect the immobilized ligand Allowing the baseline to stabilize for 30 min is recommended to obtain high quality kinetic analysis For the NLC chip it is recommended to inject 1 M NaCl in the stabilization step 5 2 5 Analyte Injection In the ProteOn XPR36 system ligands and analytes are typically injected at perpendicular directions in the 6 x 6 configuration to perform the interaction analysis This patented One shot Kinetics approach allows up to 36 individual interactions to be performed simultaneously in a single analyte injection providing high efficiency in experiment optimization and high throughput in data production Ligands and analytes are typically injected in the vertical and horizontal directions respectively For kinetic analysis analyte injections are usually performed at a high flow rate for example 100 ul min but a lower flow rate may be used to reduce sample consumption The injection conditions including association and dissociation time flow rate and analyte concentrations should be optimized to obtain high quality interaction analysis Refer to Chapter 4 section 4 3 for further
150. ns between six different d TG ligand densities with five concentrations 800 nM 100 nM 33 nM 11 nM 3 7 nM of NC were tested RU response units Interaction Analysis of Pt Squares to G Quadruplexes Zheng X et al 2012 Platinum squares with high selectivity and affinity for human telomeric G quadruplexes Chem Commun 48 7607 7609 Two new Pt squares with quinoxaline bridges selectively stabilize human telomeric G quadruplexes with high binding constants 107 109 M and an unprecedented binding stoichiometric ratio of Pt square G quadruplex 6 1 Zheng et al 2012 The binding affinity is measured using the parallel SPR analysis in the ProteOn XPR36 system 48 Applications 3 3 5 Lipid Membrane and Membrane Edri M et al 2013 Novel liposome capture surface chemistries Protein Analysis to analyze drug lipid interaction using the ProteOn XPR36 system l l Bio Rad Bulletin 6449 The ProteOn XPR36 system allows for interaction en Luo R et al 2013 Novel Liposome Capture Surface Chemistries to analysis of lipid bilayer membranes or membrane Analyze Drug Lipid Interaction Using the ProteOnTM XPR36 System proteins with other biomolecules Membrane proteins Poster presented at AAPS San Antonio USA Nov 2013 encapsulated in liposomes or lipoparticles may be immobilized to sensor chips to investigate the interactions In addition because of its flexibility for experiment design the ProteOn XPR36 s
151. nsport limitations assumes a 1 1 binding model as is the case with the simple Langmuir model but it takes into account the rate at which analyte is brought from the bulk solution to the sensor chip surface which is governed by mass transport Some biomolecular interactions may be mass transport limited if the rate of association is faster than the rate at which analyte diffuses to the sensor chip surface The following equation describes Langmuir binding with mass transport limitations kK K A pulk Te ae B lt AB t d Equation 4 where k is the mass transport rate constant for the diffusion of analyte A from the bulk solution to the surface A good test of whether an interaction is mass transport limited is to run the experiment at different flow rates and calculate the association rate constant Diffusion to the surface of the sensor chip will be faster at higher flow rates thus if the association rate of a given interaction increases with higher flow rates and decreases with low flow rates most likely the interaction is mass transport limited Usually one can get around a mass transport limited interaction by running the ProteOn XPR386 system at high flow rates or by using low ligand density however there are certain situations when even these adjustments cannot eliminate the mass transport effect and modeling the interaction using a Langmuir model with mass transport limitations is more attractive Bivalent Analyte Mo
152. o broadened the scope of SPR technology 3 5 1 Vaccine Characterization The compatibility with clinical samples enables the ProteOn XPR36 system to provide both concentration analysis and kinetic profiling of active components in clinical samples Therefore it is an efficient tool in the evaluation of vaccines for immunogenicity analysis and potency determination Published Applications Vaccine Immunogenicity Analysis Khurana S et al 2010 Properly folded bacterially expressed H1N1 hemagglutinin globular head and ectodomain vaccines protect ferrets against H1N1 pandemic influenza virus PLoS ONE 5 e11548 Khurana S et al 2010 Vaccines with MF59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian H5N1 influenza virus Sci Transl Med 2 15ra5 These articles describe how SPR technology provided by the ProteOn system was used to facilitate vaccine production against pandemic diseases For example in the first article the H1N1 A California 07 2009 virus was the target A recombinant protein approach was used to achieve rapid large scale production of vaccines The ProteOn system was used as a quality control tool H1N1 HA 1 330 and H1N1 HA 1 480 were expressed in E coli under controlled redox refolding conditions H1N1 HAO was a mammalian cell derived recombinant full length H1N1 HA virus The ProteOn system was used to verify the expression of conformational native antigenic epitopes
153. o reduce the mass transport effect while minimizing sample consumption Step Details Step Type Injection reference Blank Analyte Analyte Orientation Horizontal Note The injection parameters of analyte samples may be customized and those of the injection reference should be the same for correct referencing 7 Regeneration This phase regenerates the chip surface with ligand or capture reagent Step Details Step Type Regenerate Orientation Horizontal Note Conditions should be optimized for each interaction Regeneration should remove all the bound analyte but not damage the ligand 113 ProteOn XPR36 Experimental Design and Application Guide 7 2 Running an Experiment B The software will automatically go to the Protocol tab Select Configuration Edit the protocol name with the ProteOn XPR36 System sample container and other information as needed 7 2 1 Instrument Preparation C Select Samples and input the information of all the 1 Start the instrument Skip this step if the instrument is in continuous use A Press the power button on the left side of the instrument to the 1 position to turn on the instrument D Select Steps In the Protocol Editor panel drag steps one by one from the left side In the Step samples and reagents in the corresponding sample wells and Volume B Turn on the user computer Details panel click the arrow in the sample source C Wait until all five instrument LEDs
154. odynamics and Energetics Knowing the thermodynamics of biomolecular interactions in addition to the kinetics leads to a better understanding of the mechanisms behind binding and helps improve rational drug design SPR technology is capable of measuring the energetics of a biomolecular interaction by both equilibrium based and kinetic based analyses The typical experiment of thermodynamics involves measuring equilibrium constants under different temperatures and calculating the dissociation constant Kp at each temperature The experimental results are plotted with the Van t Hoff equation to obtain the enthalpy entropy and free energy changes of the interaction When transition state thermodynamics or energetics are desired kinetic constants are measured at different temperatures and plotted with the Eyring equation It is a useful method for understanding detailed characteristics of biomolecular interactions Because of the parallel injection feature the ProteOn XPR36 system allows for high quality and high throughput experimental results at different temperatures and greatly enhances the performance of the SPR technology in obtaining thermodynamics of biomolecular interactions The ProteOn XPR36 system s benefits include Highly efficient thermodynamic analysis workflow for structural biology Stable performance and high reproducibility transition state Q628A K660A unbound Energy Binding coordinate
155. of 100 ul min to reduce this possibility After the stabilization injection is complete look for a stable baseline If the baseline drifts a second stabilization injection may be needed or a harsher buffer may be used Part 2 Capture of Ligand For some applications such as antibody screening or capturing proteins using tags immobilization of the ligand of interest directly to the chip surface may not be desired In such a case selective capture of the ligand from a crude sample for subsequent analysis with an analyte may be preferred Major Steps for Ligand Capture For a customized ligand capture chip surface 1 Conditioning of sensor chip conditioning is performed following the sensor chip initialization process using ProteOn regeneration solutions refer to Chapter 5 2 2 Immobilization of capture reagent or biomolecule activation immobilization and deactivation using a general use sensor chips such as GLC GLM and GLH chips If working with biotinylated molecules or histidine tagged proteins NLC HTG or HTE chips may be used and this step may be skipped 3 Injection of solution containing ligand to be captured such as crude supernatant or tissue culture lysate 4 Removal of nonspecifically captured biomolecules and stabilization of ligand capture prior to interaction analysis 4 2 6 Ligand Capture Using Capture Proteins Antibody Screening In some cases capture of the ligand of interest from a cr
156. of SPR analysis making this technology ideal for use in several stages of the drug develooment pipeline With the rise of antibody therapeutics there is a need for fast and accurate determination of the affinity of candidate antibodies to their targets SPR can be used for both quantitative large and small molecule screening and qualitative relative ranking of antibody therapeutics binding or no binding during drug development Small molecules can also be screened for activity and desirable adsorption distribution metabolism and excretion ADME properties using the ProteOn XPR36 system This ability enables the identification of undesirable compounds earlier in the drug discovery process before considerable time and effort are invested in costly clinical trials 3 2 1 Antibody Kinetic Screening The ProteOn XPR36 system is ideal for screening antibody antigen interactions The applications in antibody engineering include kinetic screening epitope mapping and epitope binding Epitope mapping is a process by which epitopes on an antigen are pinpointed and with SPR technology site directed mutants of an antigen against a given antibody are screened The ProteOn XPR amp 6 system offers a distinct advantage over other SPR platforms by facilitating One shot Kinetics the rapid kinetic screening of antibody targets in a single analyte antigen injection The ProteOn XPR36 system can obtain kinetic constants of single antibody antigen intera
157. of biomolecular interactions can be used to correlate to a particular property of the analyte or the ligand or to assess cross reactivity to different biomolecules Screening antibody targets against antigens with site directed mutations is used in epitope mapping that leads to improved antibody design Competitive binding assays such as the classic sandwich assay are used to determine if different antibody molecules recognize the same or different epitopes of an antigen a process called epitope binding 2 1 7a 7b va 49 2m 1 1 Antibodies 1 and 2 Second Antibody 25 wo Antibodies 7a and 7b Flexible experimental configuration Available for various types of assays Fig 3 5 Table of epitope binding High throughput screening Published Applications Epitope Binding and Mapping Abdiche YN et al 2009 Exploring blocking assays using Octet ProteOn and Biacore biosensors Anal Biochem 386 172 180 This article Abdiche et al 2009 describes the use of the ProteOn XPR36 system to perform epitope binding using competitive binding assays Three different assays with different molecular orientations called in tandem blocking premix blocking and classical sandwich assays were performed The results from the three assays showed strong consistency When comparing among different label free biosensors the ProteOn XPR36 system also showed strong consistency with other platforms utili
158. on two experiments were performed to show the possibility of analyzing non adherent mammalian cells using the same method Figure 3 31 140 120 100 80 60 40 20 0 1 aa a Ra a E 20 Response RU 200 O 200 400 600 800 Time sec Ligand rabbit IgG Analyte Staphylococcus aureus Dilution ratio A1 x10 A2 x30 A3 x90 A4 x270 A5 x810 Flow rate 100 ul min Fig 3 31 Kinetic analysis of the IgG Fc fragment Staphylococcus aureus interaction 3 3 7 Regenerable Biotin Capture Surface Ligand capture method is a strategy often employed in an SPR experiment to increase the data quality and the reusability of sensor chips To apply the ligand capture method it is required that an affinity tag is linked to the ligand in order to facilitate the binding between the ligand and the capture reagent Widely used affinity tags include biotin histidine tag polyhistidines etc Biotin is particularly preferred in some cases due to the ease of biotinylation to biomolecules and its high affinity to the avidin family proteins such as avidin streptavidin and NeutrAvidin However the high affinity between biotin and streptavidin also results in difficulty of surface regeneration A new method of biotin based ligand capture with surface regeneration capability is possible This new method takes two affinity tags biotin and histidine tag to build a multi layer ligand immobiliza
159. on parameters Volume Flow Rate Injection Reagent Orientation ul ul min 1x EDC 1 sulfo NHS Vertical 150 30 2 4 6 mM alkylamine Vertical 150 30 3 T atmanoamnipe gt ical 150 30 HCI Note 1x EDC sulfo NHS contains a 1 1 mixture of EDC and sulfo NHS These components are included with the ProteOn amine coupling kit It is recommended to follow the instructions shipped with the kit to prepare the activation reagents for this protocol The final concentrations of the activation reagents are 20 mM EDC and 5 mM sulfo NHS 93 ProteOn XPR36 Experimental Design and Application Guide Partial Surface Modification The extent of surface modification can be used to control the liposome capture level and configuration Lower surface modification will reduce the capture capacity and in many cases improve the resistance to nonspecific binding Lower modification will also increase the chances of capturing intact liposomes while higher modification will increase the tendency of liposomes to deform and even open into lipid bilayers spread over the surface Partial surface modification refers to varying the immobilization level of undecylamine by controlling the immobilization conditions This can be achieved by controlling the activation level undecylamine concentration or injection volume Otherwise mixing undecylamine with ethanolamine HCI the deactivation solution from the amine coupling kit is an easy and efficient a
160. on spot to select a color The sensorgram appearance functions in ProteOn Manager software are shown in Figure 4 22 19 ProteOn XPR36 Experimental Design and Application Guide 4 5 7 Quality Standards for SPR Results The following standards are used to judge the quality of SPR results An example of high quality SPR results is shown in Figure 4 23 as a reference for applying the standards Visual inspection the lines of the resulting fit should pass through the experimental sensorgrams Both the fitted and original data should be displayed for publication Parameter results fitted parameters should be within an expected and reasonable range The Rmax value should be within the range of a few hundred RU ideally less than 200 RU to minimize the impact of mass transport effects ProteOn Manager software provides the choices of global grouped or local sensorgram fitting When available it is recommended to compare the results of global and grouped analyses to demonstrate the reliability of the sensorgram fitting If it is possible to perform both kinetic and equilibrium analyses on the same dataset the calculated Kp value obtained from the equilibrium analysis should be similar to the Kp value calculated from the individual k and ky values obtained from the corresponding kinetic analysis These two comparisons are usually applied to determine the confidence level of the fitted parameters The fitted parameters must be
161. quality required Which sensor chips are compatible with running buffer containing free amine salts such as Tris HCl All sensor chips are compatible with running buffer with free amine salts However avoid using these salts when immobilizing a ligand by amine coupling What is the mass transport effect and how can it be minimized The mass transport effect refers to the conditions where the transport rate or accessibility of the analyte to the ligand on the surface is restrained Thus the kinetic constant measurement is affected by the transport rate This effect is typically due to fast on rate and or very high surface density of the ligand Common solutions include reducing the ligand surface density and or increasing the analyte injection flow rate It should be noted that the mass transport effect is not a problem if its influence is insignificant to the data fitting Normally in SPR experiments biochemical factors such as ligand immobilization chemistry or analyte sample preparation have more influence on the accuracy of SPR analysis To quickly check for the mass transport effect 1 Inject the analyte at different flow rates If the same k is measured at all flow rates there is no influence from the mass transport effect But if the k decreases with decreasing flow rates the system is mass transport limited 2 Analyze data first with the Langmuir model and then with the Langmuir with mass transport model If the same k
162. r 6 full kinetics 86 data points surface regeneration not required Conventional serial flow SPR system 3 5 hr 3 full kinetics 18 data points surface regeneration required Kinetic Screening Captured Ligand Screening for mAb supernatants ProteOn XPR36 system 11 hr 96 full kinetics 576 data points Conventional serial flow SPR system 65 hr 96 full kinetics 576 data points Analyte Screening ProteOn XPR36 system 25 hr 96 x 6 full kinetics 3 456 data points Conventional serial flow SPR system 5 7 days 96 x 3 full kinetics 1 728 data points Multiplex Screening ProteOn XPR36 system 0 7 hr 6 x 6 binding matrix 36 data points Conventional serial flow SPR system 2 5 hr 3 X 6 binding matrix 18 data points Array Screening ProteOn XPR36 system 12 hr 36 x 36 binding matrix 1 296 data points Conventional serial flow SPR system no equivalent 15 ProteOn XPR36 Experimental Design and Application Guide High Data Quality Four Factors of the a D ata Processin ProteOn XPR36 System for High Quality SPR J Results Data Referencing The key step in data processing is data referencing SER ey ster Data referencing corrects for the artifacts in SPR Sufficient Signal to Noise Ratio experimental results a g The ProteOn XPR36 system has two novel PAR advantageous referencing modes that no other SPR system provides an interspot reference to correct for refracti
163. r high throughput sample processing Thus the ProteOn XPR amp 6 system is positioned as an optimal SPR biosensor platform for high data quality and cost effective experiments Ligand Immobilization Sensorgram Deactivation Activation Ligand RU Ligand Immobilization lt gt gt Time Raw Interaction Sensorgram bai D Analyte Injection _ E gt Time Processed Interaction Sensorgram R T Data Processing 2 T Time SPR Result Data Analysis gt ka Ky Kp Concentration Fig 4 1 SPR workflow for biomolecular interaction analysis RU response units In order to provide a user friendly guide for the use of the ProteOn XPR36 system the steps of data processing and data analysis using ProteOn Manager software are outlined in section 4 7 and the options for exporting SPR results for presentation are described in section 4 8 4 1 2 Checklist of Good Publication Standards Because implementing biomolecular interaction analysis with SPR biosensors incorporates a multiple step workflow all the factors of data acquisition processing and analysis should be considered upfront in experimental design An excellent resource regarding the factors to consider is the checklist of good publication standards developed by the key opinion leaders in the SPR community These standards help present SPR results in a clear and organized manner for publications facilitating effective info
164. r s disease using detrimental effects thus it represents a new generation the ProteOn XPR36 system The interaction between of therapeutic agents for Alzeheimer s disease AB assemblies and cellular prion protein PrP was AB Oligomers uM AB p Monomer 0 5 1 2 4 PBST S E SE E E pHa m o ERTIES IREIRIRIRIRIE o I RIRIRIRIRIeE creuer gt PRT a A a a a a a rest gt FR RRIRIRIRIe rest gt FRITTERS 100 x 100 100 80 5 80 5 D D gt D gt T ge l E T eo l g g g 8 l g l g l 8 40 5 8 8 40 20 l 20 i akal J inject wash SK Sale T O 200 400 600 800 O 200 400 600 800 O 200 400 600 800 Time sec Time sec Time sec Fig 3 18 Interaction analysis between PrP fragments PrP 3 230 N1 terminus C1 terminus and AB assemblies 4 uM AB oligomers a 2 UM AB oligomers Y 1 UM AB oligomers e 0 5 UM AB oligomers e RU response units 43 ProteOn XPR36 Experimental Design and Application Guide Characterization of the Interaction Between Proteins and Phosphoinositides Sommer L 2012 Identification and characterization of novel phosphoinositide binding proteins using SPR Bio Rad ProteOn Webinar Series Sommer L 2012 Using DNA hybridisation to capture liposomes for SPR based detection of phosphoinositide protein interactions Poster presented at PEGS Bo
165. raction partner onto a sensing surface and the analyte injection step refers to the injection of an analyte the second interaction partner in a flow to interact with the ligand These two steps are the data generating process in an SPR experiment Thus together they are termed data acquisition The data collected are presented as a time trace to form a sensorgram Data acquisition processing and analysis form the data flow leading to SPR results Data processing includes sensorgram processing and sensorgram referencing where referencing is the main task Data analysis includes parameter setting followed by sensorgram fitting for kinetic analysis or value plotting for equilibrium analysis and concentration analysis to yield SPR results 4 1 1 ProteOn XPR36 System The ProteOn XPR36 system is an SPR biosensor platform consisting of the ProteOn XPR36 instrument and ProteOn Manager software The system features a novel 6 x 6 experimental configuration for multiplexed interactions among multiple targets and analytes This unique patented design facilitates the simultaneous investigation of 36 different interactions using the One shot Kinetics approach The ProteOn XPR36 system facilitates the generation of high quality SPR results by providing versatility in experimental design reproducible instrument performance comprehensive referencing options and a powerful software user interface In addition this platform allows fo
166. range but as the analyte concentration is increased it approaches the theoretical maximum response R max 16 Experimental Design the limiting value When performing an equilibrium analysis use data in which the responses of all analyte concentrations have reached equilibrium and confine the fitted region to the areas where the responses are flat Note that in ProteOn Manager software the equilibrium analysis also presents the choices of fitted or constant and global or grouped for parameter setting The definitions are the same as those described in the orevious section RmaxlA oe Kp tlAl Equilibrium Equation 4 RU Baseline Time Fig 4 16 An idealized sensorgram displaying the equilibrium phase RU Dt Say A a _ For A gt gt Kp Reg R Roq A For A lt lt Kp gt R PralAl D Fig 4 17 Determination of the equilibrium constant 4 5 3 Concentration Analysis Although SPR biosensors can be used to determine analyte concentrations at binding equilibrium in a manner similar to an enzyme linked immunosorbent assay ELISA concentration analysis in SPR biosensors is usually implemented in a different approach for higher efficiency and convenience Here the initial binding rate of a biomolecular interaction is measured under mass transport limited conditions in which the binding rate is directly proportional to th
167. ray approach of the ProteOn XPR36 system to attain high throughput results Approximately 36 unique domains were immobilized to a sensor chip surface and qualitative binding results were determined against the same 36 domains injected as analytes across the surface A few interactions were discovered between different membrane protein extracellular domains and specific research on the membrane proteins containing these extracellular domains 3 3 6 Cell Antibody Interaction Analysis A new application of the ProteOn XPR36 system is the investigation of whole cell protein interactions Mammalian or bacterial cells are captured on sensor chips immobilized with cell specific antibodies allowing for the interaction of these cells with different proteins Published Applications Analysis of Cell Antibody Interactions Adachi S et al 2011 Interaction analysis of cell protein using surface plasmon resonance SPR method Medical Science Digest Japanese 37 33 38 This article Adachi et al 2011 employs the ProteOn XPR36 system to identify the binding of bacterial cells to a sensor chip surface Staphylococcus aureus bacteria expressing a high level of protein A on their surfaces were prepared in a single cell Suspension The cells have a diameter of approximately 1 um An IgG surface was used to successfully capture bacterial cells from the suspension through the high affinity binding between Protein A and the IgG Fe fragment In additi
168. re reagent or immobilizing ligands to the surface allows for the direct analysis of molecules from crude samples without prior sample purification The figure illustrates the protein quantitation capability of the ProteOn XPR36 system Figure 3 7 Clone 5 90 70 4 i T eter Ba Saa da n aAa TANAN A AA Ania a Laana MAI A 3 50 4 p paa aaah A a arean a haard adhana A Q 30 i iMi n 2 vis Tipteerers an as 10 4 EEPE NAT E NEN A WENE NEE EOE EATE YT 10 T T T O 1 2 3 4 5 Time min Clone 9 IgM in supernatant 110 tele Agatha P AR NN Vy he peop Staten fh ra y Ariaan oC A Jane Cc O Q N 00 cr 10 T T T T 0 1 2 3 4 Time min Clone 7 115 MAAD AAA Aa cone i 2 07 seme tte teatime g e ieena ea 8 40 i e 15 vr sade iterate AP esc et a aaia ey 10 T T T T T O 1 2 3 4 5 Time min Concentration Analysis 40 D 30 o N S 20 S _ N 0 cr 10 gt O O 4 8 12 16 20 24 28 Concentration nM Concentration Analysis 20 18 Standards 16 a 14 4 D 12 Control O P 10 4 cr 8 Unknown 6 samples 4 E 6 4 7 2 8 8 8 9 6 10 4 11 2 12 12 8 13 6 Concentration nM Fig 3 7 Protein quantitation capability of the ProteOn XPR36 system Applications In a typical antibody production workflow it is necessary to monitor both concentration and kinetics of antibody samples Utilizin
169. recorded when publishing SPR results Chi Chi is the average of the squared residuals the average of the squared differences between the measured data points and the corresponding fitted values The lowest value that can be expected is the baseline noise The Chi value should also be published as it indicates the fitting confidence Empirically these values should be less than 10 of R max regardless of units Residuals a plot of the residuals should form a random scattering of the same order of magnitude as the noise level It is helpful to display the residual data along with the fitted data when publishing your work Standard errors standard errors determine how sensitive the sensorgram fitting is to changes in the parameters and should be included in publications Signal to noise ratio the responses in both the association and dissociation phases must show an adequate signal to noise ratio SNR typically over 3 Given that the baseline noise is 1 RU in the ProteOn XPR36 system the sensorgram rise in the association phase and fall in the dissociation phase must be greater than 3 RU to guarantee the quality of the SPR results Response RU 120 160 200 240 280 Time sec ka ka Ky Ky Kp Rmax Chi 1 Ms Error 1 Ms 1 s Error 1 s M RU RU 179x 107 209 10 4 00 10 2 64 10 223x 10 375 2 66 Fig 4 23 An example of high quality SPR results Sensorgram
170. rformed prior to the analyte injection Real time double referencing is unique to the ProteOn XPR3 amp 6 system This method employs a blank buffer injection in parallel with the analyte injection Compared with the traditional injection reference the real time double reference has the advantages of accurate monitoring of possible changes on ligand surfaces and saving time by eliminating the additional blank buffer injection The accurate monitoring of ligand surfaces greatly enhances the referencing quality especially in the cases of capture surfaces where reversible capture of the ligand is employed and exponential baseline decay is often observed 71 ProteOn XPR36 Experimental Design and Application Guide E EA Excluded volume correction EVC is not an E independent referencing option but rather a calibration Reference Subtracted with a blank surface reference This calibration is applied Zane LEDENE a m when a cosolvent with a high refractive index such as E a DMSO is used in an analyte solution to increase the 5 7 analyte solubility A high refractive index cosolvent may ARR i Te produce a larger bulk effect on a reference surface than i Ranh Buffer Injection J on an interaction surface due to the volume exclusion of Z _ i pauni oe the cosolvent by the ligand on the interaction surface This inconsistency can be resolved by the EVC calibration i Please refer to section 4 6
171. ride side chain of the Fc region varies in different cell lines and even in different batches which compels biopharmaceutical manufacturers to analyze Ab Fc receptor binding affinity during the discovery and manufacturing phases Figure 3 36 The ProteOn XPR36 system has been used as an essential tool for biosimilar assessment in Ab Fc receptor binding affinity analysis A Light chain or A N N N Angen Fab Fab binding Biological o activity Fc region y C C B a1 6 Asn Fig 3 36 Human antibodies and their variable oligosaccharide side chains A structure of ahuman antibody depicting the amino terminal N carboxy terminal C heavy and light chain variable domains V and V respectively heavy and light chain constant domains C and C respectively disulfide bonds S S and oligosaccharide side chains CHO B example of an oligosaccharide side chain with the following components m N acetylglucosamine m bisecting N acetylglucosamine e mannose e galactose sialic acid 4 fucose 3 5 Biomedical Applications Recently the uses of the ProteOn XPR36 system have extended to a number of biomedical applications which were not typically performed by the SPR community including vaccine evaluation and clinical diagnostics These new applications have benefited from the usability and experimental convenience of the ProteOn XPR36 system They have als
172. rmation sharing among researchers using SPR biosensors This checklist The Bare Minimum Requirements for an Article Describing Optical Biosensor Experiments TBMRFAADOBE is presented here Rich and Myszka 2011 We highly recommend consulting this checklist prior to submitting SPR results for publication 60 Experimental Design TBMRFAADOBE nstrument used in analysis Identity source MW of ligand and analyte Surface type Immobilization condition Ligand density Experimental buffers Experimental temperatures Analyte concentrations Regeneration condition Figure of binding responses with fit Overlay of replicate analyses Model used to fit the data Binding constants with standard errors Reference Rich RL and Myszka DG 2011 Survey of the 2009 commercial optical biosensor literature J Mol Recognit 24 892 914 61 ProteOn XPR36 Experimental Design and Application Guide 4 2 Guide to Ligand Immobilization on the ProteOn XPR36 System SPR has revolutionized the study of biomolecular interaction by providing a platform that does not require the ligand or analyte to be labeled SPR measures the interaction between a ligand immobilized to the surface of a sensor chip and an analyte in solution This measurement takes place in real time providing kinetic equilibrium and concentration data Performing interaction analysis on an active and stable ligand surface is key to generating
173. roughput of the ProteOn biosensor in antibody screening applications Bio Rad ProteOn Webinar 2011 Series BioRadiations 133 One Array 36 Unique Protein Interactions October 2011 These articles Abdiche et al 2011 and BioRadiations 2011 and the webinar Lindquist 2011 describe in detail how to create a 36 ligand array for antibody screening Figure 3 3 This novel use of the ProteOn XPR36 system enables the immobilization of 36 individual ligands to the array surface In this assay 36 antibody targets to the same antigen were analyzed using the classical sandwich method and both epitope binding A5 AG Fig 3 3 A 36 Ligand array for high throughput screening and mapping were performed Figure 3 4 In the sandwich assay an antibody is assessed as to whether or not it can bind a preformed antigen antibody complex and form a sandwich on the surface of a sensor chip Formation of a sandwich means that the tested antibody recognizes a separate epitope than the immobilized antibody and the absence of a sandwich means that the tested antibody recognizes the same epitope as the immobilized antibody All these experiments were achieved on a single ProteOn sensor chip in approximately one day 260 240 200 160 1204 so f Response RU og O 80 160 240 320 400 480 560 640 720 800 880 960 Concentration nM Fig 3 4 36 equilibrium binding isotherms generated from dose response anal
174. s of three repeated analyte injections are overlaid and fitted together using the Langmuir model top table of fitting results bottom If the same experiment is repeated multiple times the standard deviations of the fitting results should also be shown Note The steps of data processing and data analysis using ProteOn Manager software are outlined in section 4 7 80 Experimental Design 4 6 How to Perform Excluded Volume For example prepare 4 6 DMSO EVC standards 5 DMSO running buffer and 5 DMSO stock Correction on the ProteOn XPR36 analyte from 10x PBS buffer as shown in Table 4 4 Protein Interaction System Dilute the stock analyte solution using the fresh 5 In experiments where analytes are dissolved in a DMSO interaction running buffer Figure 4 25 cosolvent with a high refractive index such as DMSO Table 4 4 Preparation of DMSO solutions Analyte and DMSO running the reference surface produces a larger bulk solvent buffer concentrations are given as an example and may change according response than the ligand surface Normally this bulk to individual experimental needs effect can be cancelled out after reference subtraction o ml edie ml ade ml ie ml u However the bulk effect is not equal on the interaction TE a T ar T and reference spots The reference surface produces DMSO 0 4 0 6 100 0 08 a larger bulk shift effect because of the larger an 8 6 8 4 1 700 oo Nalyte i concentration
175. se was immobilized to approximately 24 000 RU and CBS was injected in a threefold dilution series ranging from 20 0 082 uM RU response units 16 000 E Conventional layer NHS T E Conventional layer sulfo NHS 12 000 4 ProteOn layer NHS 3 E ProteOn layer sulfo NHS c 2 8 0007 Oo N 8 4 000 7 E O Standard capacity chip type chip type High capacity Fig 2 5 Comparative coupling efficiency Representative data for immobilization of rabbit IgG Ligand coupling efficiency of the ProteOn chip s easily activated layers is higher than in conventional layers and activation of ProteOn chip layers is higher using sulfo NHS instead of NHS RU response units 22 ProteOn Sensor Chips Table 2 1 Representative immobilization efficiencies on ProteOn sensor chip surfaces designed for high protein binding capacity Non Bio Rad GLM Chip GLM Chip GLH Chip Chip NHS NHS Sulfo NHS Sulfo NHS Activation Activation Activation Activation Protein pl RU RU RU RU Pepsin 3 70 750 2 050 2 470 Ovalbumin 4 5 2 800 3 400 6 700 6 800 Protein A 51 4 300 3 500 6 000 18 800 B2 microglobulin 5 3 2 600 3 250 3 650 12 400 Carbonic anhydrase ll 5 9 6 600 2 300 6 000 9 000 21 200 Myoglobin 6 9 7 4 3 900 2 800 7 000 12 200 Polyclonal IgG 6 8 10 000 9 700 12 200 22 200 2 3 2 ProteOn Sensor Chips for Site Specific HTE and HTG Sensor Chips Immobilization Attachment NLC HTG and HTE of Histidin
176. se the ProteOn XPR36 system has an inline degasser However samples should be degassed if they contain air bubbles How do I prevent evaporation of samples when working with microplates Should I cover the plate with a simple seal Please use the sealing film that is provided with the ProteOn XPR3 amp 6 system for microplates Additional film can be ordered from Bio Rad Laboratories have experiment protocols from sensor chips coated with a carboxylated dextran layer Can I apply them on ProteOn sensor chips that are coated with a carboxylated alginate layer Yes In rare cases some protocols may need to be slightly adjusted due to the different surface chemistry The carboxylated dextran surface is a highly charged polymer which may be difficult to activate and require high concentrations of activation reagents This high surface charge can cause nonspecific binding of some analytes The carboxylated alginate surface of ProteOn sensor chips is easily activated and has a low charge density Compared to the carboxylated dextran surface the carboxylated alginate surface usually requires fewer activation reagents 108 Frequently Asked Questions Why does the ProteOn XPR36 system intentionally create separation air bubbles during sample uptake Separation air bubbles are intentionally created between the sample and the running buffer to prevent mixing You may choose how many bubbles are generated based on the injection
177. se two major steps should be taken into consideration The subsequent data processing and analysis should also be taken into consideration during experimental design Please refer to Chapter 4 for details For an SPR experiment how do estimate the theoretical maximum analyte ligand interaction response R_ First measure the ligand immobilization response R from the ligand step Second use the equation below to calculate theoretical maximum analyte ligand interaction response R _ M n R max L L R n stoichiometric number of the analyte ligand interaction M analyte molecular weight M ligand molecular weight For example if an antibody ligand of 150 kD is immobilized to 1 000 RU an antigen analyte is 30 kD and the interaction ratio is 1 1 The Rmax is calculated as follows R 1 x 30 150 x 1 000 200 RU There are two channel referencing and two double referencing options What do they mean and which should I use for my experiment Channel referencing is the minimum referencing required for SPR analysis Double referencing is the secondary referencing that is used with the primary referencing in certain applications such as ligand capture surfaces Figure 6 5 106 Frequently Asked Questions SPR Reference Primary 1 Reference aN Secondary 2 Reference Channel Reference Double Reference Option 2 Option 2 Option 1 Option 1 Interspot Reference Injection Reference
178. ss microfluidics It enables the creation of a 6 x 6 interaction array on a sensor chip This guide is written for the regular experiment layout in the ProteOn XPR36 system which includes injecting ligands in vertical channels and analytes in horizontal channels A ProteOn Manager software protocol for an experiment consists of seven basic phases as shown below Setting optional Conditioning Immobilization Stabilization EVC calibration for applications with DMSO containing running buffer nteraction Regeneration optional 1 Setting This phase ensures the system is ready to perform the experiment It is used to set the chip temperature flush the system with running buffer and allow time for the system to come to thermal equilibrium after instrument startup Step Details Step Type Set Temperature Set Buffer 2 Conditioning This phase prepares the chip surface for use It is optional but highly recommended because it increases baseline stability The protocols of conditioning are listed as follows Note For the LCP chip used in the ProteOn liposome capturing kit conditioning is performed after the biotin ssDNA capture step and before the liposome capture step Step Details GLC GLM and GLH Chips Step Reagent Orientation Volume Flow Rate 1 0 5 SDS Horizontal 30 ul 30 ul min 2 50 mM NaOH Horizontal 30 ul 30 ul min 3 100 mM HCI Horizontal 30 ul 30 ul min 4 0 5 SDS Vertical 30 u
179. ston USA Apr 2012 Phosphoinositides are important lipid signaling molecules found in all cellular membranes and also in a non membranous endo nuclear form within the nucleus Phosphoinositide signaling is mediated through interaction with proteins that contain specific phosphoinositide binding domains The ProteOn XPR36 system was used to analyze some phosphoinositide protein interactions of interest Figure 3 19 GST 2xFYVE HRS 250 nM PC _ 0 2 mg ml ce PIZP 2x PI5P gt 6007 PI94 5 P F J 2x PI 4 5 P 5 400 7 PRF Q N oO ca 200 O 200 100 O 100 200 300 400 500 Time sec Interaction between FYVE motif of HRS and endosomal PI3P GST PH PLC61 125 nM 2 000 PCo 2 mgm PI 1 600 2x PI 4 5 P D gt P 4 5 P r 2x PC oO 2 rs 0 2 mg ml g 12007 __ 513 45 O F amp 8004 400 4 O 200 100 O 100 200 300 400 500 Time sec Interaction between PH domain of PLC61 and PI 4 5 P Fig 3 19 Interaction of phosphoinositides with various proteins containing phosphoinositide binding domains and proteins that lack a phosphoinositide binding domain RU response units Lekomtsev S et al 2012 Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis Nature 492 276 279 The article _ekomtsev et al 2012 describes the research on the molecular m
180. t 4 C To avoid condensation on the chip surface which can lead to inaccurate results keep sensor chips in the sealed nitrogen filled pouch during storage Sensor chips should also remain in the pouch until reaching room temperature before use Temperature equilibration takes from 30 to 60 min Opening a Sensor Chip 1 After temperature equilibration cut the top seal of the aluminum pouch 2 Hold the black end of the cartridge up inside the pouch to ensure the sensor chip slide remains inside the cartridge 3 Press the sensor chip slide firmly into place within the cartridge 4 Remove the sensor chip cartridge from the aluminum pouch Sensor chip slide Cartridge Initializing a Sensor Chip New sensor chips must be initialized in the instrument the first time they are used in an experiment Follow these steps to initialize the sensor chip using either air or glycerol 90 Tips and Techniques 1 Insert the temperature equilibrated sensor chip 5 2 Running Experiments into the instrument chip loader The chip ID chip 2 with Sensor Chips chemistry and chip expiration date populate the Chip Details area of the Sensor Chip box in An interaction analysis experiment comprises five major steps ProteOn Manager software 1 Conditioning 2 Choose one of the initialization options using either a air or glycerol Ligand immobilization 3 The additional Use Last initialization option is Stabili
181. t samples were used to show the workflow of quantikinetics and verify its high performance Figure 3 9 K Ka A bulk Purace B AB K Ka d AB kA dt Fig 3 8 SPR technology for concentration analysis Immobilize anti mouse IgG Ab Quantify and capture Ab from supernatant Inject Ag for kinetic analysis Fig 3 9 Workflow of quantikinetics 3 2 4 Drug Compound Screening An important step in small molecule drug discovery is the screening of large libraries of small molecules for affinity and activity against a protein target SPR technology is extremely valuable in small molecule drug discovery because it provides information on activity and specificity that allows for the quantitative ranking of lead compounds The ProteOn XPR36 system has very good sensitivity that allows for the detection of molecules as small as 100 Da High capacity sensor chips such as the GLH and HITE chips are ready for analyzing small molecules since their high surface capacity for ligand immobilization increases the binding response for a given analyte In addition the high throughput and the versatility in experimental design facilitated by the novel 6 x 6 interaction array allows for the production of high quality SPR results using the ProteOn XPR36 ystem High sensitivity gt 95 Da High throughput screening Available for fragment screening Published Applications Screening of Carbonic Anhydrase Inhibitors Bravman T et a
182. t with the chip surface The excited surface plasmons are very sensitive to the refractive index change at the surface of the thin metal film Thus the incident angle of the light required for SPR is impacted by the refractive index change of the molecules in contact with the chip surface In an SPR binding experiment this refractive index change is brought about by binding of analyte in solution to ligand immobilized on the chip surface therefore tracking the change in the incident angle required for SPR allows one to monitor biomolecular interactions in real time The change of the incident angle required for SPR is defined as SPR response in the unit of response unit RU 1 RU is 1 1 000 000 of 1 refractive index unit and is roughly equivalent to a surface density of protein at approximately 1 pg mm For a more in depth discussion of SPR see a recent review that offers an overview of SPR theory and different SPR configurations Daghestani et al 2010 This reference describes the theory and application of a number of optical biosensors including surface plasmon resonance biosensors and the different configurations for each Plotting the SPR response over time during the interaction between an analyte and a ligand results in a sensorgram A sensorgram is a visual presentation of the interaction Figure 1 1 illustrates the terms for a Sensorgram using for instance an antibody antigen interaction The binding response initially incr
183. tion configuration It incorporates the advantages of both affinity tags the regeneration capability of the histidine tag surface chemistry and the high affinity biotin surface chemistry Published Applications Proof of Principle for Regenerable Biotin Capture Surface Zhu M et al 2018 A novel biotinylated ligand capture method with surface regeneration capability for label free biomolecular interaction analysis BioRadiations Feb 2013 This article Zhu et al 2013 describes the workflow of using both HTG and HTE chips to achieve regenerable biotin capture surface As the proof of principle two steps were implemented in an SPR experiment 1 histidine tagged streptavidin was captured to the chip surface through the interaction between the histidine tag and the activated tris NTA on the chip surface and 2 a biotinylated ligand was captured to the streptavidin to prepare the ligand surface for interaction analysis In this approach the histidine tagged streptavidin is the primary capture reagent and the activated tris NTA is the secondary Figure 3 32 When it comes to surface regeneration a chelate such as EDTA is typically used to break the binding between histidine tag and tris NTA and the original surface with only tris NTA is restored Excellent results were shown for this approach 52 Applications 15t capture reagent 20d capture reagent 20d capture reagent Capture Chip surface gt Chip surface Regener
184. tion models based on the Langmuir equations Langmuir with drift and Langmuir with mass transport Langmuir with drift is commonly used in experiments that use a capture surface for example the reversible antibody or histidine tag capture surface In such cases the captured ligand may escape from the capture reagent on the chip surface leading to baseline drift before the analyte injection and during the association and dissociation phases Note that this model calculates only a linear drift that is constant with time Blank buffer referencing should be used to gain accuracy when correcting for a large baseline drift showing an exponential curvature as described in section 4 4 The second model is Langmuir with mass transport Mass transport is the process whereby an analyte diffuses from the bulk solution to the chip surface To determine whether a particular interaction is limited by mass transport and thus whether this model should be used inject an analyte sample at different flow rates If the association curves are different then this interaction is mass transport limited In contrast if the association curves are independent of the flow rate all binding curves overlap then diffusion is not the rate limiting factor and the simple Langmuir model can be applied Other Binding Models There are four complex binding models for analyzing non Langmuir interactions the heterogeneous analyte heterogeneous ligand two state and bi
185. tion solution Regenerate Orientation Horizontal 5 EVC Calibration This phase accounts for excluded volume effect Follow Chapter 4 section 4 6 for setting up the steps Calibration is needed when DMSO is used as cosolvent in sample and running buffer in small molecule application Step Details Step Type EVC Calibration Orientation Horizontal 6 Interaction This phase analyzes the interaction between the ligand and the analyte The following factors should be considered 1 Prepare the analyte samples in the running buffer to form a dilution series typically a two or threefold dilution series centered at the expected K value 2 When needed set up a double reference Replace one of the six analyte channels with running buffer for use as a real time double reference row reference Alternatively set up an injection of running buffer to all six analyte channels prior to the injection of analyte samples injection reference Double referencing is needed to correct for baseline drift and is used mostly when the ligand is captured reversibly by a capture reagent such as antibody NeutrAvidin NLC and LCP chips or tris NTA complex HTG and HTE chips 3 Ensure that the contact time of the interaction is long enough to observe curvature in the association phase 4 Ensure that the dissociation time of the interaction is long enough to observe adequate signal drop in the dissociation phase 5 Optimize flow rate t
186. tiple surfaces for different assays Unattended running for assay validation Typical assay optimization involves screening different experimental conditions for ligand immobilization and analyte injection including ligand density immobilization OH analyte concentration ionic strength choice of additives etc The combination of different parameters results in a large matrix of experimental conditions for optimization Utilizing the novel 6 x 6 interaction array the ProteOn XPR36 system has the power to achieve an entire optimization experiment in a short period of time and provides the highest efficiency and accuracy compared to other SPR platforms available Optimization advantages include 6 x 6 experiment conditions in an injection Efficient data analysis by the software Published Applications Design and Optimization of Antibody Analysis Assays Bronner V et al 2009 Rapid screening and selection of optimal antibody capture agents using the ProteOn XPR36 protein interaction array system Bio Rad Bulletin 5820 This technical note Bronner et al 2009 describes how the One shot Kinetics approach was used to rapidly screen the binding of four antibody binding proteins and seven types of antibody targets The selection of antibody binding proteins that provide the optimal binding characteristics for the capture of each antibody type was achieved rapidly in the ProteOn XPR36 system The One shot Kinetics approach allows for
187. to account for the excluded volume effect while maintaining the accuracy of referencing Refer to Chapter 4 section 4 6 for details on how to apply this correction Troubleshooting Analyte Injection Nonspecific Binding Nonspecific binding NSB is defined as the direct binding of an analyte or sample components other than the target ligand to the sensor chip surface NSB is characterized by significant binding responses that occur on reference spots and do not return to baseline at the end of the injection Figure 5 3 These events can potentially skew experimental results In theory if the NSB responses on the interaction surface and on the reference are similar subtracting the reference from the interaction response will correct the data and lead to accurate fitting to the binding model In practice however it is very difficult to determine whether NSB is similar on the interaction and reference surfaces There are cases where the ligand molecules on the interaction surface block NSB on the chip surface This leads to a higher NSB response on the reference surface and results in incorrectly referenced data or even negative responses 96 Tips and Techniques At present NSB is one of the most difficult factors to optimize in label free biomolecular interaction analysis There are two main strategies used in SPR biosensors to overcome NSB 1 Using a reference surface that is as similar to the ligand surface as possible Optima
188. ts are immobilized to the sensor chip surface followed by a single orthogonal injection of six unique analytes In addition to traditional kinetic measurements SPR technology can be used to qualitatively assess biomolecular interaction properties monitor the quality and or concentration of biologics and investigate binding thermodynamics B C Fig 3 1 Generation of the 6 x 6 ligand analyte interaction array A six ligands are immobilized in six parallel ligand channels B six analyte samples are injected into six analyte channels orthogonal to the six ligand channels C detail of a single ligand analyte interaction spot green showing the positions of the two interspot references yellow The versatility of the microfluidics in the ProteOn XPR36 system makes it an ideal complement to the drug discovery and development workflow for target screening and characterization studies as well as assay design and optimization The ProteOn XPR36 system is capable of monitoring up to 36 unique biomolecular interactions simultaneously in a variety of configurations depending on the experimental design The ProteOn XPR36 system can also be used in downstream processes such as protein quantitation for quality control as SPR can be used to monitor the concentration of small molecules and biologics in crude or impure samples 3 2 Large and Small Molecule Screening The versatility of the ProteOn XPR36 platform expands the capabilities
189. ude sample such as a hybridoma supernatant or phage display supernatant prior to analysis with an analyte may be the method of choice This type of noncovalent capture is ideally suited to the ProteOn XPR36 system as the 6 x 6 array allows for the rapid screening of hundreds of antibodies Ligand Capture Conditions for mAb Screening To capture a mAb from a crude hybridoma supernatant create a sensor chip that contains a relevant capture protein to capture the mAb such as an anti IgG antibody or protein A G These anti lgG and protein A G surfaces can be created using the direct amine coupling method described previously Using R nax to Determine Ligand Capture Conditions for mAb Screening Consideration must be taken to ensure that enough of the mAb is captured to be able to interact with its analyte The level of mAb captured is dependent on the amount of mAb available in the supernatant and on the efficacy and immobilization level of the capture protein In this case the R nax equation must be used twice First determine how much mAb must be captured to be able to see an analyte response of 200 RU Second calculate how much of the protein capture anti IgG or orotein A G would need to be immobilized to attain the required mAb level 4 2 7 Ligand Capture by Biotin Label or Histidine Tag The NLC HTG and HTE Sensor Chip The NLC chip allows for the selective capture of ligands that contain a biotin tag such as proteins DNA
190. um capacity amine coupling for protein protein and protein small molecule interactions GLH High capacity amine coupling for protein small molecule interactions NLC NeutrAvidin for biotinylated molecule capture HTG Compact capacity tris NTA for histidine tagged large molecule capture HTE High capacity tris NTA for histidine tagged small molecule capture LCP Used with the LCP capturing reagent kit for liposome capture Fig 2 1 An overview of the seven different types of ProteOn sensor chips GLC GLM GLH NLC HTG HTE and LCP with the specific application for each chip listed 2 3 1 Amine Coupling ProteOn Sensor Chips GLC GLM and GLH Three ProteOn sensor chips are available for amine coupling The GLC GLM and GLH chips provide for compact medium and high ligand surface capacities respectively All three of these chips are functionalized with easily activated carboxylic acid groups that can be reacted with the activation reagents EDC and sulfo NHS to react specifically with free surface amines of proteins Bio Rad offers the amine coupling kit for use with the GLC GLM and GLH sensor chips GLC Sensor Chip Compact Binding Capacity The GLC sensor chip is designed with an extremely thin alginate layer for amine coupling of protein ligands at a compact 6 KRU surface capacity The compact structure of the alginate layer helps mitigate mass transport effects that are more often observed with thicker layers of surface
191. urface of kinetic binding model allows for the calculation of a sensor chip and then flowing an analyte an interacting kinetic parameters such as the association rate biomolecule such as another protein or a small molecule constant k in the unit of M s the dissociation over the chip surface to investigate the binding affinity rate constant k in the unit of s and the and binding kinetics between the analyte and the ligand equilibrium constant K in the unit of M K A B AB K q Biomolecular interaction in a new light Two biomolecules A and B interact with each other to form a complex AB Using an SPR biosensor along with the equilibrium constant K the association rate constant k and the dissociation rate constant k can be measured determining more details of the interaction compared to other methods Introduction Advantages of the ProteOn XPR36 System The ProteOn XPR36 protein interaction array system is The parallel flow SPR biosensor platform an SPR biosensor platform that provides real time label free analysis of the specificity affinity and kinetics of biomolecular interactions Using the XPR36 configuration the system features a novel 6 x 6 interaction array for the simultaneous analysis of up to six ligands with up to six analytes The unique design increases the versatility of Screens multiple panels of analytes experimental design and the productivity of experimental Acquires the resonance an
192. valent analyte models When choosing a model to determine the binding kinetics of interactions the Langmuir or Langmuir with mass transport model should be selected by default since the majority of biological interactions occur in a 1 1 stoichiometry It is necessary to provide a biological justification for the use of other models and conclusions based on analyses with these complex models should be confirmed with additional experiments Langmuir Model with Drift The Langmuir model with drift is used when a biomolecular interaction follows simple 1 1 binding but exhibits a persistent baseline drift that interferes with data interpretation This is applied in SPR experiments using capture agents as the captured ligand may leach from the surface over time The Langmuir model with drift uses the same kinetic equations as the simple Langmiur model but calculates the drift as a linear drift with time D t where D is the slope of the drift It should be noted that this model should be applied to the experiments with slow baseline drift because fast baseline drift caused by the rapid decay of the captured ligand usually shows an exponential curvature and does not fit with this model The optimal solution is correcting the baseline by the subtraction of a blank buffer reference reference of blank analyte buffer over ligand surface 14 Experimental Design Langmuir Model with Mass Transport Limitations The Langmuir model with mass tra
193. values are obtained there is no influence from the mass transport effect If the k is lower with Langmuir analysis then the system is mass transport limited When and why do I apply excluded volume correction Refer to Chapter 4 section 4 6 In experiments where analytes are dissolved in a cosolvent with a high refractive index such as DMSO the reference surface produces a larger bulk solvent response than the ligand surface because of the larger concentration of cosolvent near the reference surface It is due to the exclusion of cosolvent by the ligand near the ligand surface This difference in bulk effect causes inaccurate reference subtraction Excluded volume correction uses a dilution series of DMSO solutions to correct for the difference of bulk effect between interaction and reference surfaces 109 ProteOn XPR36 Experimental Design and Application Guide Quick Guides CHAPTER 7 he quick guides outline the workflows for writing an experimental protocol and running an experiment on the ProteOn XPR36 system ProteOn XPR36 Experimental Design and Application Guide 7 1 Writing a ProteOn XPR36 Experiment Protocol Surface plasmon resonance SPR is a biosensor technology that measures biomolecular interactions in a real time and label free manner The ProteOn XPR36 protein interaction array system is an SPR platform that utilizes the novel technology XPRS6 to enable parallel flow channels and crisscro
194. ve index change bulk effect and nonspecific binding and a real time injection reference to correct for baseline drift resulting from the changes in the ligand Response RU D 4 l 50 0 50 100 150 200 surface Time sec l Note For additional information about the referencing options in Fig 1 5 ProteOn XPR36 system signal to noise ratio ProteOn the ProteOn XPR36 system watch the video at www bio rad com XPRS6 system noise is 1 RU and 2 RU after double referencing SPR proteon reference responses over three times signal to noise ratio 8 x SNR are detectable RU response units A Baseline drift 6 ligand injections Instrument Stability J l l l l l 240 2004 160 o 2 ES O 2 120 29 O c o 2 80 oO 2 wo S T P Baseline corrected T T T RE 2 200 O 500 1 000 1 500 2 000 2 500 3 000 3 500 Q Time sec ac Fig 1 6 Evaluation of kd value reproducibility using the ProteOn marpo One shot Kinetics kit 2 systems x 3 chips x 6 ligand channels x 6 analyte channels 216 sensorgrams CV 6 1 over 2 systems and 6 sensor chips RU response units 2 Experiment Design n ProteOn XPR36 System Configuration Optimizes Multiple 4 4 Analyte Factors Simultaneously Vertical channels Ligand Optimize ligand immobilization Capture reagent Surface chemistry P g Ligand conditions Horizontal channels J J J J l l PPE optimize analyte injection Ligand is
195. ves the highest level of immobilization For example BSA has a pl of approximately 5 5 To have a positive charge the protein must be dissolved in a buffer of pH less than 5 5 Therefore one might wish to try a series of buffers with a pH of 5 5 5 0 4 5 and 4 0 and monitor which results in the highest level of immobilization Care must be taken to ensure that the immobilization conditions used result in an immobilized ligand that retains its activity When using buffers of extreme pH the ligand may be denatured or unfolded and therefore lose its activity see Table 4 1 Detergents may also be added into the immobilization buffer but salt should be kept to a minimum just enough to keep the ligand soluble Ligand Injection Parameters Determining the ideal ligand injection parameters is Important Flow rate and contact time can have significant effects on immobilization Default injection parameters are 30 ul min for 5 min The low flow rate will help to increase immobilization as will an increase in the injection contact time Ligand Conditions The concentration of the ligand will also affect the total amount immobilized Typically concentrations of 5 100 ug ml should be sufficient to attain a good level of immobilization The ligand stock buffer should have a high concentration of the ligand so that when it is diluted with the immobilization buffer any salts or other additives oresent in the stock buffer will also be diluted
196. vironment that prevents denaturation of the immobilized ligand and nonspecific adsorption of the analyte The increased surface area of the 3 D structure of the alginate layer provides more attachment sites than would a completely flat surface and results in more ligand molecules being immobilized on the chip surface In addition the molecular weight and structure of the alginate coating can be modified to create sensor chips with different surface capacities This results in sensitive detection of interactions with minimal surface effects on binding The surface chemistry of the ProteOn sensor chips allows the ProteOn XPR36 system to detect tight binding interactions down to picomolar concentrations of analytes or analytes as small as 95 Da The ProteOn sensor chips combined with the unique 6 x 6 interaction array allow for the interaction of up to 36 separate ligand analyte pairs on a single chip increasing the throughput of a single experiment The ProteOn sensor chips are based on innovative patent protected surface chemistry The general use ProteOn sensor chips GLC GLM and GLH sensor chips come functionalized with carboxyl groups to facilitate amine coupling of protein ligands via surface exposed amine groups In addition to serving as attachment sites the carboxyl groups serve to concentrate the ligand at the surface of the sensor chip as the negatively charged carboxyl groups attract proteins rendered positively charged
197. ximab antibodies Currently quantitative infliximab testing is costly Using the ProteOn XPR36 system a quantitative assay for infliximab in serum was developed and in general the practicality of using SPR in the clinical lab was explored 57 ProteOn XPR36 Experimental Design and Application Guide CHAPTER 4 Experimental Design A surface plasmon resonance SPR experiment designed to investigate biomolecular interactions incorporates an entire workflow including data acquisition data processing and data analysis High quality SPR results can be obtained when all the steps in this workflow are designed and executed appropriately ProteOn XPR36 Experimental Design and Application Guide 4 1 Introduction to SPR Experimental Design An SPR experiment to investigate biomolecular interactions incorporates an entire workflow including data acquisition data processing and data analysis High quality SPR results can be obtained when all the steps in this workflow are designed and executed appropriately The workflow should be optimized to achieve this goal This chapter introduces the SPR workflow and provides optimization approaches to facilitate high quality SPR results Four steps are typically performed to complete an SPR experiment ligand immobilization analyte injection data processing and data analysis as shown in Figure 4 1 The ligand immobilization step refers to the immobilization of a ligand the first inte
198. xperimental Design and Application Guide 4 4 Guide to SPR Data Processing on the ProteOn XPR36 System 4 4 1 Interaction Sensorgram Terms An SPR sensorgram is a graph of time traced SPR responses during a biomolecular interaction analysis The x axis is time in seconds and the y axis is SPR response in response units RU It should be noted that both ligand immobilization and analyte injection steps generate sensorgrams but the sensorgram in the analyte injection step is more often used because it contains the ligand analyte interaction information for affinity or kinetic analysis Therefore where not otherwise specified sensorgram hereinafter refers to that generated in the analyte injection step As illustrated in Figure 4 4 an SPR sensorgram can be divided into three different phases Baseline is the phase before the injection of the analyte Running buffer flows over the sensor chip surface bearing the immobilized ligand and the baseline response is recorded Association is the phase during the injection of the analyte The analyte flows over the sensor chip surface and binding occurs between the ligand and the analyte A concave increasing response curve is produced Depending on the binding kinetics of the interaction partners and the experimental conditions the increasing response curve may or may not reach a plateau which indicates that interaction equilibrium has been reached Dissociation is the phase after the i
199. ysis of an analyte over the 36 ligand array RU response units High Affinity Antigen Antibody Interaction Analysis Votsmeier C et al 2012 Femtomolar Fab binding affinities to a protein target by alternative CDR residue co optimization strategies without phage or cell surface display mAbs 4 341 348 This article describes the affinity maturation of adalimumab using an approach that employs quantitative screening of soluble Fab fragments with diversification to complementarity determining region CDR and alternative recombination to co optimize large sets of affinity improving mutations The approach achieved 500 fold affinity improvement and resulted in the first reported femtomolar affinity antibody against protein without display screening The ProteOn XPR36 system was employed to characterize the binding kinetics and affinity between the antigen and the antibody variants The result shows the capability of the ProteOn XPR36 system to detect dissociation constants less than 1 x 10 st and equilibrium constants lt 1 pM which are the typical limits for label free biosensors in analyzing high affinity biomolecular interactions 32 Applications 3 2 2 Epitope Binding and Mapping First Antibody Qualitative assessment of binding can be used to rank the relative binding of antibody to targets map or bind epitopes and define structure activity relationships for small molecules The binding results obtained from SPR analysis
200. ystem is used as a platform for solution based membrane protein research with soluble recombinant proteins The benefits The ProteOn liposome capturing kit used within the ProteOn XPR36 system provides a novel hydrophilic surface chemistry that allows for advanced applications such as drug lipid interaction analysis Edri et al 2013 This technical note and the poster show that it is possible to establish an evaluation tool using this kit for include small molecule drug delivery including measuring the Hydrophilic surface chemistry allowing for high properties of drugs and evaluating drug carrier systems performance for capturing lipid assemblies Figure 3 29 Hydrophilic surface chemistry allowing for easy regeneration Real time referencing for reliable experimental results Published Applications Applications of Liposome Capturing Kit and GLC Lipid Kit Luo R et al 2012 Novel lipid membrane protein application kits for label free biomolecular interaction analysis Poster presented at PEGS Boston USA May 2012 The ProteOn liposome capturing kit and the ProteOn GLC lipid kit are the novel lipid membrane protein application kits designed for capturing lipid assemblies such as liposomes for lipid protein lipid small molecule and membrane protein protein interaction analysis Luo et al 2012 The kits have been used in a variety of applications that characterize lipid based biomolecular interactions including 1 anti
201. zation available for reuse of the sensor chip If the used sensor chip is taken out and reinserted in the instrument glycerol initialization must be used Analyte injection OY a ON Regeneration 5 2 1 Conditioning Conditioning prepares the chip surface for use Although optional it is highly recommended because It can increase baseline stability The conditioning protocols for all sensor chips are listed in Table 5 1 Setting Up a Protocol 1 Choose New or Open from the menu bar to open the database browser 2 Choose a Template Protocol or Experiment Edit the name as needed for your new experiment 3 In the Protocol screen edit the configuration samples and protocol steps as needed 4 In the Instrument Control screen set the chip temperature and sample temperature Table 5 1 Conditioning parameters GLC GLM and GLH Chips Injection Reagent Orientation Volume ul Flow Rate wl min 1 0 5 SDS Horizontal 30 30 2 50 mM NaOH Horizontal 30 30 3 100 mM HCI Horizontal 30 30 4 0 5 SDS Vertical 30 30 5 50 mM NaOH Vertical 30 30 6 100 mM HCI Vertical 30 30 NLC Chip Injection Reagent Orientation Volume ul Flow Rate ul min 1 50 mM NaOH Horizontal 30 30 2 1 M NaCl Horizontal 30 30 3 50 mM NaOH Vertical 30 30 4 1 M NaCl Vertical 30 30 HTG and HTE Chips Injection Reagent Orientation Volume ul Flow Rate ul min 1 0 5 SDS Horizontal 30 30 2 50 mM NaOH Horizontal 30 30 3 10
202. zed Bravman T et al 2007 Screening ranking and epitope mapping of anti human IL 9 supernatants Bio Rad Bulletin 5540 This technical note Bravman et al 2007 describes how the ProteOn XPR36 system was applied to the selection ranking and epitope mapping of 20 mAb supernatants The surface immobilized with anti mouse mAb was used to capture supernatant antibody as the ligand and the analyte IL 9 was injected to analyze the mAb Ag interactions Figure 3 5 The throughput of the system was capitalized by capturing five different ligands in parallel and determining the binding kinetic constants in a single injection of five analyte concentrations Figure 3 6 The four strong binding antibody samples were purified and then epitope mapping was performed It should be noted that mAb supernatants containing IgM were analyzed Although the kinetics could not be fitted to the simple Langmuir model it showed the possibility of qualitative analysis for IgM samples 33 ProteOn XPR36 Experimental Design and Application Guide Clone 1 115 gt 90 7 o 2 65 S 40 pene aad aed ween naman bra ne ee B pte iN OY Peres 15 eee ee painiar inaa es ake ee Ee Sancnehahitpind sag GIAN AAI EYOTA ara A OO O OON E OE NE ny 10 T i T T T O 1 2 3 4 5 Time min Clone 2 115 gt 90 P a 65 2 PT tanai aa anana diaid aids ashi i i ii il a iih a iid Lai Q 40 p Par AA DAPA YER

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