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User's Guide to Alpha Assays Protein:Protein

1. In a similar fashion the expression of Protein 2 tagged with GST could be confirmed using Anti GST Acceptor beads and the same Anti GST antibody biotinylated and captured by streptavidin Donor beads Figure 23 Note that the Anti GST antibody is polyclonal which allows a sandwich capture of the GST protein As a result the signal increases with increasing target protein concentrations The assay was performed in a 384 well OptiPlate for a 25 uL final assay volume 1 Add 10 uL Anti GST Acceptor Beads 20 ug mL final diluted in assay buffer 2 Add 5 uL CHO lysates dilutions in lysis buffer 3 Incubate 15 min at room temperature 4 Add 5 uL of biotin Anti GST 1 nM final in assay buffer 5 Incubate 30 min at room temperature 6 Add 5 uL of streptavidin Donor beads 20 ug mL final diluted in assay buffer 7 Incubate 15 min at room temperature 8 Read on an Alpha compatible reader EnVision or EnSpire Multimode Plate Readers v GST Protein 2 1 Protein 2 GST e CHO WT FFE S R5 br Cells well Bb dla Figure 23 Detection of a GST tagged protein in two transfected lysates using an Alpha assay The detection of GST tagged Protein 2 with either an N or C terminal tag was done in a sandwich assay involving Anti GST Acceptor beads and a biotinylated goat Anti GST antibody Serial dilutions of lysates were tested in the sandwich assay The amounts of lysate are reported in cells well equiva
2. 5 1 4 Cell treatment In certain conditions treatment of the cells is recommended to achieve an efficient cellular interaction between the two proteins In some cases starving the cells may constitute a treatment that will favor the desired protein protein interaction In other cases treating the cells with a known modulator of one of the proteins of interest agonist antagonist inhibitor etc might be an elegant way to generate a control cell lysate In this example these would be a negative or basal state lysate and a positive stimulated lysate It will then be possible to calculate an assay window based on the signals produced with the positive and negative lysates If no treatments are available the negative control cell lysate could be a lysate prepared from cells transtected with one protein or the other 5 1 5 Cell lysis Lysis buffers The measurement of protein protein interactions using Alpha requires the production of cell lysates to release the proteins from their cellular environment This is a critical step since the lysis per se is a disruptive process involving deter gents that could break the protein protein interaction under investigation To address that particular need various non denaturing formulations of moderate strength have been developed over the years for pull down and immuno precipitation assays The efficiency of lysis buffers to break cells and solubilise proteins will also depend on c
3. USER S GUIDE TO ALPHA ASSAYS PROTEIN PROTEIN INTERACTIONS p gt PerkinElmer For the Better Table of Contents 1 Introduction e mc mues ep m HE e 6 A bee AAA 2 2 ASSav oe i a O a a a e E ROUES ROCA ee 3 3 2 1 Bead choice EEN 3 2 1 1 AlphaLISA vs AlphaScreen 2 aa 3 2 1 2 Bead coating OPUONS s a s c ta x 4 RO ROEGRO amp OX 43 ROBORE RO BEES OER EE EOE BES 3 22 DUNE CNO ee ro ras eee 4 E AAA 8 7 25 General CONGEDI ens so e O A A a A AA 8 2 2 2 Buffer components that may interfere a a a a a 8 3 Assay development biochemical protein protein interaction assays 11 3 1 The first experiment protein cross titration 2 ll ll s 11 3 2 Cross titration when you are using antibodies to capture proteins to beads 2 2 2 14 3o Ordet OFICIO uem URGE coa or RR OX OR XR y WO ROO OX SUR P ae 15 3 4 Displacement assay assay validation ee 16 4 Determining K in a biochemical assay o oo ole 20 4 1 Saturation curves Tor determination of Kj wem on 64 x RR RS RR 6686445 ELE RA x RE we 20 2 2 Competition binding assays for determination of uuum uem ive G44 3m oom daa a4 21 5 Cell based assays 22 sss 24 5 1 Studying protein protein interactions using overexpressed tagged proteins 24 5 1 1 Selection and orientation of protein tags lee 25 5 1 2 Generation of expression Vector ue 8 ae umo um XX ow WORK BOE On ROGER X xo
4. The development of new assays and optimization of existing assays using Alpha assay technology is typically accomplished in short timeframes with relatively small investments of labor and cost Alpha assays are well suited for a multitude of R amp D applica tions and lower throughput assays but the technology is also ideal for HTS high throughput screening Following initial assay optimization at a particular final reaction volume assay volumes are easily scaled up or down without the need for re optimization or increased reagent concentrations One can Table 8 Microplates for Alpha assays and recommended volumes Catalog Color Microplate simply increase or reduce all volume additions proportionately without loss in sensitivity or assay performance Higher signal and S B ratios are often achieved with higher density micro plates see Table 8 for available microplates Using low volume assay formats in 384 well Shallow well ProxiPlates or 1536 well plates can yield significant savings in cost per well and help preserve precious and scarce reagents We recommend using a plate seal cover to prevent evaporation of samples and subsequent signal inconsistencies PerkinElmer TopSeal A PerkinElmer Cat No 6050195 is ideal for this purpose and does not interfere with the Alpha signal permitting the plate to be read without first removing the plate seal Recommended number assay volume Comments Y AreaPlate 96 600
5. and applications such as phage display ELISA and EMSA electrophoretic mobility shift assay have been adapted to Alpha 4 Avidity because each bead has multiple binding sites low affinity interactions can be detected using nanomolar concentrations of proteins or other binding partners This allows you to minimize the amount of proteins required to perform the biomolecular interaction assay 5 Ease of use the assay is homogeneous meaning that no wash steps are necessary Many types of pre coated beads are available to capture biotinylated FITC labeled DIG labeled GST tagged 6X His tagged and other tagged binding partners Protein A Protein G Protein L and anti species beads are available to capture protein to a bead using an antibody Unconjugated beads are available for direct conjugation of an antibody or other reagent of choice to a bead Custom bead conjugation and custom assay development services are also available www perkinelmer com alphatechnology 2 Assay Design 2 Assay design Please consider the following factors when measuring protein protein interactions with an Alpha assay 2 1 Bead choice 2 1 1 AlphaLISA vs AlphaScreen Both AlphaLISA and AlphaScreen technologies rely on the use of the Alpha Donor bead However this Donor bead can be paired with either an AlphaLISA Acceptor bead or an AlphaScreen Acceptor bead The main difference between these two Acceptor beads is the final fluorophore used to g
6. because other components in the cell lysate may have the ability to interact with the GSH and Ni beads causing sample interference In such cases it may be preferable to use a bead coated with anti tag antibody Additionally if you have purified your GST tagged proteins or His tagged proteins using an affinity column and will be using a GSH or Ni bead in your Alpha assay you will need to dialyze away any glutathione or imidazole in your purified protein preparation These components will interfere with the interaction between the tagged protein and the bead Cross titration MEK1 unactive binding to ERK2 unactive MEK1 unactive Hook point x 0 1 nM 200 000 SNM 300 000 10 nM 30 nM 100 nM 300 nM 100 000 9 8 7 log ERK2 unactive M Figure 6 Low affinity interaction on higher capacity beads illustrating a higher bead capacity as depicted by a higher hooking point A cross titration was performed to characterize the interaction between His tagged MEK1 and GST tagged ERK2 Glutathione GSH Donor beads and nickel chelate AlphaLISA Acceptor beads were used to capture the proteins and generate the assay signal The expected K for this interaction is 29 uM Bardwell A J et al 2001 A hook point is reached at 100 nM GST ERK2 and between 100 nM and 300 nM His MEK1 Assay Design 2 2 Buffer choice The information that follows is based on experiments performed using the AlphaScreen TruHits beads T
7. 7 Add 5 uL of streptavidin Donor Beads 20 ug mL final diluted in assay buffer 8 Incubate 60 min at room temperature 9 Read on an Alpha compatible reader Envision or EnSpire Multimode Plate Readers Once a positive Alpha assay signal is validated users can refer to the AlphaLISA Assay Development Guide from PerkinElmer for subsequent steps 0 L T T T T 8 5 8 0 7 5 70 6 5 6 0 log recombinant Protein 2 M Figure 25 Competition assay performed to validate a protein protein interaction assay for Protein 1 FLAG and Protein 2 GST Lysates prepared from CHO cells double transfected with Protein 1 FLAG and Protein 2 GST DNA were tested in the presence of increasing concentrations of recombinant Protein 2 An IC value of 73 nM was calculated www perkinelmer com alphatechnology 30 Cell based Assays 5 2 Studying protein protein interactions using endogenous proteins Protein expression assessment using antibody pair specific for each target Alpha assay Western Blot Section 5 2 4 Alpha assay matrix experiment to test interaction Identification Preparation of cell lysates using various lysis buffers Section 5 2 3 Antibody selection amp reagent preparation Sections 5 2 1 5 2 2 of complex antibody pair and optimal lysis buffer Section 5 2 5 31 Positive signal confirmation in Alpha assay Sect
8. Alpha technology The tolerance of Alpha technology to various detergents and other substances is detailed in section 2 2 2 Confirmation that a positive signal is a valid protein protein interaction Once the double transfections have been performed at various DNA ratios and cell lysates have been prepared the validity of the protein protein interaction assay should be confirmed One or more of the following approaches may prove useful in validating that a positive Alpha assay signal from double transfected cell lysates is indeed due to the desired protein protein interaction e As negative controls test single transfected lysates in the protein protein interaction assay e To confirm that the signal observed correlates with the amount of double transfected cell lysate perform a titration of the amount of double transfected lysates added to the assay Figure 24 e To confirm that the signal observed is due to the presence of the two specific proteins when possible perform a competition experiment in which increasing concentrations of a recombinant form of one of the two proteins is added to the cell lysates Figure 25 e To further confirm the specificity of the Alpha assay signal reference compounds or peptides known to interfere or modulate the protein protein interaction can be tested for their effect on the assay Titration of double transfected cell lysates in Alpha assays A cell lysate titration experiment should b
9. Anti GST antibody GST fusion proteins and peptides 3nM Biotinylated GST with streptavidin bead Anti 6X His antibody His tagged proteins and peptides 100 nM 6X His GST with glutathione beads Anti FLAG antibody FLAG tagged proteins and peptides 100 nM Biotinylated FLAG with streptavidin bead Anti maltose binding protein MBP antibody MBP tagged proteins 1 nM Biotinylated MBP with streptavidin beads Anti HA antibody Hemagluttinin tagged proteins and peptides 10 nM Biotinylated PEG HA with streptavidin bead Anti c myc antibody C myc tagged proteins and peptides 100 nM Biotinylated c myc with streptavidin bead Anti DIG antibody Digoxigenin labeled proteins peptides oligos etc 1 nM Biotinylated digoxigenin with streptavidin bead Anti FITC antibody FITC or fluorescein labeled proteins peptides oligos sugars small molecules etc gt 1 nM Biotinylated ERE FITC with streptavidin bead Anti V5 antibody V5 tagged proteins targets 3 nM Biotin Chromalink V5 14 aa with streptavidin beads Anti GFP antibody GFP tagged green fluorescent protein tagged proteins and peptides 3 nM Biotinylated GFP with streptavidin beads Glutathione GSH GST fusion proteins and peptides 300 nM 1 uM 6X His tagged GST with nickel chelate bead Nickel chelate Ni His tagged proteins and peptides 300 nM 1 uM 6X His tagged GST with glutathione bead Protein A Antibodies 3 nM antibody Biotinylated rabbit IgG with streptavidin bead Protein G Antibodies 1 nM antibody Biotinyla
10. Biotinylated Analyte Anti Analyte conjugated Alpha Acceptor Bead 929 m9 291 93279 29 29 EXCESS ANALYTE CONCENTRATION All binding ses oocuped Maximam signal production Bead association inhibited Figure 3 Illustration of the hook effect in a protein detection assay Hooking can occur on either the Donor beads or the Acceptor beads The hooking effect is sometimes masked in Alpha protein protein interaction assays resulting in what can look like a traditional saturation curve that reaches a plateau rather than hooking Figure 5 In this case two competing equilibria are occurring the signal may be decreasing because of the hooking effect on the bead but the protein protein interaction may still be increasing because the equilibrium of the protein protein interaction is being driven to more protein protein complex with higher concentrations of protein You will want to choose protein concentrations below the hook point for your assay In any case it is useful to have a rough idea of the binding capacity of the various beads particularly if you are studying a weak K gt 100 nM interaction Cross titration biotin EGF binding to EGFR Fc 500 000 4 Hook point 100 nM EGFR Fc 30 nM EGFR Fc 10 nM EGFR Fc 3nM EGFR Fc 1nM EGFR Fc 0 3 nM EGFR Fc 0 1 nM EGFR Fc 0 nM EGFR Fc 400 000 300 000 200 000 H z q 100 000 log biotin EGF M Figure 4 High affinity protein protein interaction illustrating the ho
11. GST coated Acceptor beads biotinylated p53 and GST tagged HDM2 Here are a few advantages that the Alpha technology offers over other technologies in the study of protein protein Interactions 1 Distance because the singlet oxygen can travel up to 200 nm or more in solution the distance between the Donor and Acceptor beads can be very large and still generate signal This allows the measurement of very large proteins and other large complexes In contrast traditional FRET fluorescence resonance energy transfer requires that the two FRET fluorophores be within 9 nm of each other for efficient energy transfer Moreover in FRET the energy transfer occurs through nonradiative dipole dipole coupling meaning that orientation is also an important parameter In Alpha assays the energy transfer between the two beads is chemically mediated via singlet oxygen 2 Broad range of affinities Alpha can detect a broad range of affinities with dissociation constants K ranging from picomolar to low millimolar Because the assay is homogeneous no wash steps are required transient interactions can be measured 3 Variety of interactions Alpha has been used to study a wide variety of interactions including protein protein protein peptide protein DNA protein RNA protein carbohydrate protein small molecule receptor ligand and nuclear receptor ligand interactions Both cell based and biochemical interactions have been monitored
12. adding the beads one at a time It is also possible to perform a one step assay where all of the assay components proteins beads are added to the well at the same time Figure 12 The effect of these protocol changes will need to be determined experimentally for each assay As a note for proteins or antibodies that are biotinylated at multiple positions it is generally recommended to add the streptavidin coated bead last This is because a protein that is biotinylated at multiple positions could bridge two streptavidin coated Donor beads before any other interac tion can occur as the streptavidin biotin interaction occurs quickly in comparison to other interactions This would likely make the biotinylated protein unavailable for any further interaction leading to a false negative result Additionally pre incubating each protein with its associating bead is usually not recommended as the bead will slow down the rotation of the protein in solution This could slow down the kinetics of the protein protein interaction requiring longer incubation times Also the bead may create steric hindrance preventing the protein from binding to its partner protein at all Pre incubation of a bead with an antibody that will eventually be associating with one of the proteins is sometimes performed Assay Development Add Protein X and Protein Y Add Protein X and Protein Y Add Protein X Protein Y Donor beads 4 4 and Acceptor beads Incubate
13. an Alpha assay to capture each individual protein with specific antibodies or by Western blot Alpha cell based protein protein interaction protocol refer to Figures 28 or 29 for plate map as applicable 1 Add 10 uL of lysates in a white 96 well 7 AreaPlate 2 Add 15 uL Acceptor beads 20 ug ml final assay conc diluted in assay buffer 3 Incubate 30 min at room temperature 4 f applicable Add 10 uL Biotinylated Anti tag antibody 1 nM final diluted in assay buffer 5 Incubate 60 min at room temperature 6 Add 15 uL streptavidin Donor beads 20 ug mL final assay conc diluted in assay buffer 7 Incubate 60 min at room temperature 8 Read in Alpha compatible reader EnVision Multilabel Plate Reader or EnSpire Multimode Plate Reader For endogenous protein protein interaction events the first experiment will involve evaluating lysis buffer and antibody selection We recommend using 20 000 cells well as the lysate volume Antibody pair 1 2 3 4 5 6 y 8 9 10 11 12 Pair 1 Pair 2 Lysis buffer A Lysis buffer B Pair 3 Pair 44 Pair 1 mi U07 rn u 2o Pair 2 Pair 3 Lys s b ffe e Lys is b uffer D Cells well lysates 1 2 3 4 5 61 8 9 10 11 12 20 000 A 10 000 B Lysis buffer A Lysis buffer B 5 000 C 0 D 20 000 E 10 000 F Lysis buffer C Lysis buffer D 5 000 G 0 H Cell lysate Neg Control Figure 28 First e
14. beads 5 mg mL 245 uL assay buffer 6 During first incubation Prepare a 5X working solution 100 ug mL of Donor beads keeping protected from light 5 uL Donor beads 5 mg mL 245 uL assay buffer Assay Development B Displacement protocol for a 96 well 7 AreaPlate total assay volume of 50 uL Refer to the plate map Figure 15 You can use a multi channel repeat pipettor to quickly dispense reagents into the plate with a TopSeal A adhesive seal or lid to prevent evaporation Add 10 uL Acceptor beads and 10 uL Donor beads to each well final conc 20 ug mL each Incubate 60 min at room temperature in dark place in a drawer with a TopSeal A adhesive seal or lid to prevent evaporation Read on an EnVision or EnSpire www perkinelmer com alphatechnology 18 Assay Development e Samples are set up in triplicate note that wells D1 F1 are only used to provide more wells so that O nM untagged Protein X can be measured These particular wells should not have 100 uM untagged Protein X untagged untagged untagged untagged Protein X Protein X Protein X Protein X untagged Protein X Figure 15 96 well plate map for displacement experiment untagged untagged untagged untagged untagged untagged untagged untagged Protein X Protein X Protein X Protein X Protein X Protein X Determining K in a Biochemical Assay 4 Determining K in a biochemical assay The K of a protein prote
15. cells expressing each individual protein before working with lysates from cells coexpressing the two binding partners 5 4 Cell based references When the proteins to be studied do not include any tag the confirmation of expression and selection of the best lysis buffer can be made using techniques such as Western blotting It is also possible to create Alpha assays for detecting each protein individually by using two specific antibodies in a sandwich assay format Performing these validation assays in an Alpha assay format may require some additional time and effort initially but will ultimately save hands on time as compared to Western blotting These benefits can prove especially useful when handling a large number of experimental conditions e g optimization of transfected DNA amounts for each protein See Section 5 1 7 for help with setting up the Alpha protein protein interaction assay Literature examples of Alpha cell based assays using recombinant and tagged proteins Reference Tags used Lysis buffer Lysis protocol Assay buffer Lysate amount Becker et al Virology 2008 FLAG and HA 100 mM Tris pH 8 0 100 mM NaCl 0 5 NP 40 0 2 mM PMSF containing Roche complete protease inhibitor Twenty four hours post transfection cells from one well of a 6 well microplate were rinsed with PBS and lysed on ice for 5 minutes The cellular extracts were then PBS 0 1 BSA 2 uL of lysate from one well was di
16. coating options A variety of standard Alpha Donor and Acceptor beads are offered as regular catalog items Table 1 The type of bead coating selected will most likely depend on whether the proteins being studied are available biotinylated tagged expressed as GST fusion proteins 6X His tagged proteins etc or otherwise labeled FITC fluorescein labeled digoxi genin labeled If the protein is unlabeled you could use an antibody directed against the protein to capture the protein onto a bead It is also possible to directly conjugate a protein antibody DNA oligo lectin etc to an uncoated bead Uncoated beads are coated with a dextran aldehyde These aldehyde groups can be easily covalently attached to NH groups in any protein or peptide through reductive amination The reaction is simple and detailed protocols are available Other chemistries can also be used If you have some flexibility regarding the tagged or labeled state of your proteins you may want to consider bead capacity and sample type when choosing a bead Bead capacity In a protein protein interaction the amount of protein protein complex formed is dependent on the concentrations of each binding partner within the well Increasing the concentration of either protein will push the equilibrium to more protein protein complex A B SAB A B lt Tag Each type of Alpha bead has a characteristic bead capacity Table 2 This is the point at which the beads ar
17. depletion protein depletion meaning that the percentage of bound protein can be above 1096 In this case the amount of free ligand protein cannot be estimated as being equal to total ligand protein for the curve fitting equa tion If the expected K is not below the binding capacity of the beads used in the assay a competition binding assay must be used to determine the K for the protein protein interaction The reason for this is that the bead system will become saturated when the binding capacity is reached and two competing equilibria will be occurring excess protein will be dissociating the interaction between the two beads decreasing signal the hooking effect while the excess protein may simultaneously be driving the interaction between the two proteins increasing signal The curve generated will be a composite of these two equilibria once the binding capacity has been reached This composite curve may appear to reach saturation may appear to begin to hook decrease slightly or may continue increasing without reaching saturation depending on the actual K the concentrations of proteins being tested and the binding capacities of the beads used in the assay Symptoms that a saturation curve cannot be used to accurately predict the K 1 Underestimated K in Alpha assay If the Alpha data is giving a K that is below what is expected for the protein protein interaction it is possible that bead saturation i
18. fluorimeters and luminometers cannot read Alpha assays e We recommend preparing only what you need for the day s experiments Do not store working dilutions of beads for more than one day e The Donor beads used in Alpha assays are somewhat light sensitive We recommend working under subdued lighting conditions when working with the beads less than 100 Lux the level of light produced on an overcast day Incubate the plate in the dark for example placing the covered or sealed plate in a drawer Biochemical Assays In this initial experiment you will be keeping the concentrations of beads constant 20 ug mL final concentration of each bead and varying only the concentration of each protein in a 40 pL final assay volume reaction The plate map for this Add 10 uL Protein X to each well final assay conc 0 nM 300 nM Add 10 uL Protein Y to each well final assay conc 0 nM 300 nM Incubate 60 min at desired temperature Add 10 uL Acceptor beads to each well final assay conc 20 ug mL Incubate 60 min at room temperature in dark Add 10 uL Donor beads to each well final assay conc 20 ug mL Incubate 60 min at room temperature in dark Read on an Alpha compatible reader EnVision Multilabel Plate Reader or EnSpire Multimode Plate Reader e The Alpha signal is temperature dependent If you will be performing incubations at 37 C or other temperatures we recommend that you equilibrate the plate back to room
19. occur The data are presented two different ways as a 3 D bar graph generated using Micro SOft Excel and as a titration curve plotted using GraphPad Prism Figure 9 A hook point is reached at 10 nM biotinylated EGF and 3 nM EGFR Fc after which point the signal begins to decrease Figure 10 shows an example using MEK1 and ERK2 You will most likely want to choose a pair of concentrations that give good S B while conserving protein If you will be deriving affinity constants in later experiments you will need to choose concentrations of proteins that are below the bead capacity refer to section 4 for more information 3 2 Cross titration when you are using antibodies to capture proteins to beads If you will be using antibodies to capture your proteins to the Donor and or Acceptor beads you will also need to choose a concentration of antibody to use in the assay For most assays a final antibody concentration of 1 nM 3 nM or 10 nM will usually give good results Refer to Figure 11 for a sample plate map Depending on the affinity of your antibody for the protein you may be able to titrate the protein concentration higher than described above if the antibody has weak affinity for the protein Cross titration MEK1 unactive binding to ERK2 unactive 300 000 MEK1 unactive 0 1 nM 3 nM 10 nM 30 nM 100 nM 300 nM 200 000 0 10 9 8 7 log ERK2 unactive M Figure 10 A second example of a protein
20. protein interaction it is certainly the most important step towards that goal Examples and recommendations using Alpha assays are given below but other methods may also be used see next section Tag based detection with Alpha technology Typically the confirmation of protein expression in a cell lysate can be done by performing a competition type assay set up to detect a tagged positive control peptide included in an AlphaScreen kit for example a biotinylated and flagged peptide To this assay are added increasing amounts of lysate containing the expressed protein bearing the expression tag see Figure 21 A signal decrease propor tional to the amount of cell lysate is expected as the expressed protein can compete with the positive control This step should be done for each protein If necessary the amount of DNA transfected can be adjusted to achieve the desired expression levels Non transfected cells or cells transfected with an unrelated target should be included as negative controls to rule out any interference coming from non specific components of the lysates Figure 21 Schematic diagram of an assay used to confirm cellular expression of the tagged protein The overexpressed tagged protein competes for binding of biotinylated tagged peptide to anti FLAG conjugated Acceptor beads Cell based Assays Figure 22 is an example where the expression of a FLAG tagged protein was confirmed in transfected CHO cells The assay
21. temperature before reading to ensure signal uniformity across the plate Assay configuration A variety of Alpha Donor and Acceptor beads are offered to design your protein protein interaction assay including streptavidin coated beads anti FITC and anti DIG coated beads anti fusion tag antibody coated beads nickel chelate and glutathione beads anti species antibody coated beads Protein A Protein G and Protein L coated beads Unconjugated beads are also available would you need to create your own beads Different configurations may give different results Contact PerkinElmer for more advice assay Figure 27 will be designed to test multiple possible combinations of each protein s concentration in a matrix The assay is performed in singlicate one well per condition 1 2 3 4 5 6 7 Protein X 300nM 100nM 30nM 10nM 3nM 1nM 0 3nM OnM Protein Y J A 300 nM B 100nM C 30nM D 10nM E 3nM F 1nM G 0 3 nM H 0nM O O bd O 00000000 00000000 SIBISIPLO o o Go o eeeee L4 24 2 24 4 CICAD 2 L4 2 2 28 4 COCOCOCOCOD Figure 27 96 well white Y AreaPlate map 40 uL final assay volume Next optimization steps if desired 1 Order of addition adding both beads at same time or all proteins and beads at same time etc 2 Displacement assay demonstrating that tagged Protein X or tagged Protein Y can be displaced using untagged protein 3 Incubation ti
22. transfection cells were collected in 500 mL PBS pelleted and then lysed by suspending in 20 mL lysis buffer The cellular extracts were then separated following centrifugation for 5 min at 13 000 rpm Not disclosed The cell lysates were incubated for 30 min on ice and then centrifuged at 9 357xg in a microcentrifuge for 5 min at 4 C see Section 5 1 5 not disclosed PBS 0 196 BSA PBS 0 1 BSA 100mM HEPES pH 7 5 1mM EDTA 5mM DTT 0 1 CHAPS 5 glycerol 50 mM Tris HCl pH 7 4 150 mM NaCl 0 196 BSA Forty eight hours post transfection cells were collected in 500 mL PBS pelleted and then lysed by suspending in 20 ul lysis buffer 5 ul of this cell extract were mixed to a total assay volume of 25 yl not disclosed Cells seeded in 24 well plates Forty eight hours post transfection cells were collected in 500 mL PBS pelleted and then lysed by suspending in 20 uL lysis buffer 5 uL of this cell extract were mixed to a total assay volume of 25 yL Not mentioned CHO cells grown in 6 well plate transfected and grown for 48 h then lysed in 100 uL 18 uL of this lysates is used per well of Alpha 5 ul of 4M mL lysates in 25 uL total assay volume www perkinelmer com alphatechnology 34 35 Start up Guide Quick Start Guide to Alpha Protein Protein Interactions Before you begin e Alpha assays require a special reader capable of measuring an Alpha assay Many standard time resolved
23. used in high throughput screening assays These molecules are designed to break the colloidal interactions between molecules of detergents and proteins such as BSA and thereby eliminate the presence of bubbles Bubble formation can greatly hinder liquid handling and reading The most common anti foaming agents are Antifoam A Cat No A5633 and Antifoam 204 Cat No A8311 from Sigma These agents show no interference with Alpha assays at concentrations up to 0 196 In one comparison Antifoam A was shown to be more efficient in reducing bubbles than Antifoam 204 Cofactors reducing agents and preservatives Many assays require the presence of cofactors reducing agents or preservatives in the assay buffer Alpha assays are tolerant of a wide variety of these molecules including the following categories Table 5 Category Alpha compatible additives Nucleotides up to 10 mM All nucleotides derived from adenine guanine thymine and uridine including free nucleotide mono di and triphosphate and cyclic nucleotides Reducing agents up to 10 mM Preservatives up to 0 1 Dithiothreitol DTT 2 mercaptoethanol tris 2 carboxyethyl phosphine TCEP Kathon thimerosal ProClin 300 Note Sodium azide is one of the most popular preservatives used with commercial products However it is known as a potent singlet oxygen quencher and thus it will quench Alpha signal strongly IC of 0 005 Sodium azide should be avoided
24. 0 03 Benzalkonium chloride 0 005 or less 0 03 CHAPS 0 02 or less 0 10 Chenodeoxycholate 0 002 or less 0 02 Cholate 0 002 or less 0 15 Decyl sulfate 0 196 or less 0 50 Deoxycholate 0 003 or less 0 03 Myristyl sulfobetaine 0 1 or less 0 50 Pluronic F127 0 196 or less 0 30 Sodium lauryl sulfate SDS 0 02 or less 0 2096 Benzylhexadecyldimethylammonium chloride tThese recommended concentrations have been obtained by analysis of a dose response curve using the TruHits kit Streptavidin Donor beads Biotinylated AlphaScreen Acceptor beads Assay Design Proteins and polymers Proteins and other polymers are commonly added to reduce assay background Alpha assays are tolerant of the following additives at up to 0 5 in solution e Bovine serum albumin BSA casein gelatin heparin poly lysine salmon sperm DNA Dextran T500 both from natural and synthetic sources However sources of BSA and casein have been found to vary in quality These proteins must be of a high purity to avoid the presence of contaminants that could interfere with the assay One such impurity is biotin which is often present in casein of low grade We recommend the following suppliers and catalog numbers for Alpha assays e BSA Sigma Cat No A7030 e Casein 5 solution Novagen Cat No 70955 Table 5 Compatibility with cofactors reducing agents and preserving agents Anti foaming agents Anti foaming agents are increasingly
25. 5560 white 40 50 uL The 96 well plate that is recommended for the highest sensitivity in a 50 pL reaction OptiPlate 96 6005290 white 100 uL CulturPlate 96 6005680 white 100 pl Coated for use in tissue culture OptiPlate 384 6007290 white 24 50 uL The 384 well plate that is recommended for the highest sensitivity in a 50 pL reaction AlphaPlate 384 6005350 light gray 24 50 uL Light gray color reduces potential for well to well crosstalk ProxiPlate 384 Plus 6008280 white 20 uL AlphaPlate 1536 6004350 light gray 8 10 uL Light gray color reduces potential for well to well crosstalk References References 1 Bardwell A J Flatauer L J Matsukuma K Thorner J amp Bardwell L A conserved docking site in MEKs medi ates high affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission J Biol Chem 276 10374 10386 2001 Becker M N Todd T M amp Moyer R W An Amsacta moorei entomopoxvirus ortholog of the poly A poly merase small subunit exhibits methyltransferase activity and is non essential for virus growth Virology 375 624 636 2008 Cassel J A Blass B E Reitz A B amp Pawlyk A C Development of a Novel Nonradiometric Assay for Nucleic Acid Binding to TDP 43 Suitable for High Throughput Screening Using AlphaScreen Technology J Biomol Screen 15 1099 1106 2010 Dawson R et al The N terminal domain of p53 is natively unfolded J Mol B
26. Incubate y Incubate Add Donor and Acceptor beads Add Acceptor beads 4 Read plate Incubate Incubate y Read plate Add Donor beads Incubate y Read plate Figure 12 Examples of different order of addition protocols 3 4 Displacement assay assay validation Displacement assay GST HDM2 binding to biotin p53 One way of validating the assay is to demonstrate that the displaced by unlabeled p53 association between Protein X and Protein Y can be disrupted 400 000 by adding an untagged version of one or the other protein GST HDM2 nM ICs uM Increasing concentrations of the untagged version of Protein X 300 000 o 1 0 61 or Protein Y can displace the tagged reagent from the assay 2 0 58 disrupting the association between the beads The signal did 5 0 90 should decrease with increasing concentrations of the O e 10 1 10 untagged competitor Figures 13 and 14 6 5 4 3 log p53 peptide M Figure 14 Displacement assay for biotinylated p53 GST HDM2 interaction using streptavidin Donor beads and anti GST AlphaScreen Acceptor beads Untagged 053 peptide was titrated from 30 nM to 100 uM Increasing concentrations of untagged p53 competed with biotinylated p53 for binding to GST HDM2 result ing in a decrease in signal untagged p53 cannot associate with the streptavidin Donor bead and therefore cannot generate signal Figure 13 Assay principle for displacement assay Untagged Protein
27. RGS AL134 AL101 AL102 AL126 AL104 AL105 AL103 AL106 AL107 AL132 AL130 AL131 Unconjugated 6762013 Fc specific antibody 6762003 6772001 AlphaScreen bead kits contain streptavidin Donor beads AlphaScreen Acceptor beads and a biotinylated probe 6760603 Streptavidin Donor Anti GST AlphaScreen Acceptor 6760619 Streptavidin Donor Ni chelate AlphaScreen Acceptor 6760611 Streptavidin Donor anti c myc AlphaScreen Acceptor 6760613 Streptavidin Donor anti FLAG AlphaScreen Acceptor 6760604 Streptavidin Donor anti DIG AlphaScreen Acceptor 6760612 Streptavidin Donor anti HA AlphaScreen Acceptor 6760605 Streptavidin Donor anti FITC AlphaScreen Acceptor 6760617 Streptavidin Donor Protein A Acceptor 6760607 Streptavidin Donor anti rabbit IgG AlphaScreen Acceptor 6760606 Streptavidin Donor anti mouse IgG AlphaScreen Acceptor www perkinelmer com alphatechnology 4 Assay Design Table 2 Relative capacities of various beads beads used at 20 ug mL final concentration in assay Theoretical binding capacity Molecule and partner bead Bead coating Used to bind capture provided for relative comparison used to determine theoretical binding capacity Streptavidin Biotinylated peptides proteins oligos sugars small molecules etc 30 nM Biotinylated peptide Strep Tactin Strep tag Il One STrEP tag or biotin tagged targets 100 300 nM Strep tag IL 6X His
28. The concentrations listed are the final concentrations in the presence of beads The heavy metal concentration can be higher than the value shown at initial stages of the assay so long as it is at or below the recommended level after addition of the final assay components Detergents and background reducing proteins Many assays will require the presence of additives to prevent aggregation to ensure solubility and stability of components and to reduce non specific signal background Alpha assays can tolerate the presence of a wide variety of detergents and protein additives but care must be taken in some cases Detergents The following detergents will have no negative effect on Alpha assays at up to 396 final concentration in solution un less otherwise indicated Brij 35 NSB256 Dodecane Sulfonate Tergiton NP9 EMPIGEN Triton X 100 IGEPAL CA630 o Tween 20 Guanidinium chloride Tween 80 up to 100 mM Dodecyltrimethylammonium Mowiol 4 88 bromide DTAB Nonidet P40 Octyl sulfate NSB195 NSB201 Some detergents when used at high concentration will have a significant effect on Alpha assays Table 4 This effect is most likely due to denaturation of proteins present on the surface of the beads The following table illustrates those detergents and their effects Table 4 Interference of detergents in AlphaScreen TruPoint Assay Detergent Recommended Interference levelt level IC BHDA 0 005 or less
29. X competes with GST tagged Protein X for binding to Protein Y Because untagged Protein X cannot bind to the Donor bead a signal cannot be generated from an untagged Protein X His tagged Protein Y complex As a result signal decreases with increas ing concentrations of untagged Protein X www perkinelmer com alphatechnology 16 Assay Development A Preparation of reagents 1 Prepare a 5X working solution of tagged Protein X in your assay buffer Choose a concentration based on data from your cross titration experiment 2 Prepare a 5X working solution of tagged Protein Y in your assay buffer Choose a concentration based on data from your cross titration experiment 3 Prepare a 5X working solution of untagged Protein X in your assay buffer suggested concentration of 5X solution 500 uM If you happen to know the expected K for your protein protein interaction choose a concentration that is above the K value if possible 4 Perform a serial dilution of the untagged Protein X in Eppendorf tubes as follows t may not be feasible to prepare a 500 uM solution of your untagged protein Start at the highest concentration possible f the K for your protein protein interaction is known you will want to center the final concentrations of untagged protein around the K if possible Ideally you want to cover a 3 4 log range 5 During first incubation Prepare 5x working solution 100 ug mL of Acceptor beads 5 uL Acceptor
30. as this is a potent scavenger of singlet oxygen and will inhibit the Alpha signal Proclin Sigma Aldrich Cat No 48912 U or Kathon Sigma Aldrich Cat No 48175 U is recommended as a preservative and anti microbial agent 2 2 2 Buffer components that may interfere Here are some guidelines for choosing a buffer that will allow for optimal Alpha assay readouts It is important to note that these guidelines refer to optimal conditions for the Alpha detection reagents themselves namely the Donor and Acceptor beads as well as the chemistry that leads to light emission The proteins you are working with may also have their own restrictions in terms of assay buffer composition For more support on optimal assay conditions please contact our Technical Support group to discuss your specific needs Choosing the buffer Alpha Technology is very tolerant to the types of buffering agents buffer concentrations and buffer pH used in most assays The following buffers have been tested in Alpha assays and have been shown to give excellent performance at concentrations from 10 mM to 100 mM Acetate HEPES Bis Tris MES Bis TRIS propane MOPS CAPS Phosphate Carbonate PIPES Citrate Tris Formate Buffer pH A range of pH from pH 2 5 to 9 has no influence on the performance of Alpha beads and signal generation Even pH values up to 10 5 can be accommodated but a slight loss of performance should be expected Nevertheless keep in mind that th
31. assay When performing a saturation curve to derive K it is important to choose protein concentrations above below and centered around the expected K If possible at least eight protein concentrations should be chosen ranging from 10X K to 0 1X K The concentration of only one binding partner protein should be varied the other protein should be held at a constant concentration that is below the binding capacity of its associating bead and at least 10X below the highest concentration of the varied protein Saturation binding HDM2 binding to p53 400 000 e Total Binding A Non Specific Binding 4 6 8 GST HDM2 nM Figure 17 Saturation curve for interaction between GST HDM2 and biotinylated p53 The expected K for this interaction is 0 3 UM Because the expected K is above the estimated binding capacity of the anti GST coated Acceptor beads and the streptavidin coated Donor beads a saturation curve should not be used to determine K 4 2 Competition binding assays for determination of K In many cases a competition binding assay will need to be used to determine the K in Alpha format This is because many protein protein interactions have dissociation con stants that are above the binding capacity of the beads In the competitive binding assay increasing concentrations of the untagged version of Protein X or Protein Y are used to displace the tagged reagent from the assay disrupting the association between th
32. at Preparation of Titration of various cell Positive signal l variable DNA ratios cell lysates lysates in Alpha assay O coexpression of using optimal identification of optima Ahaan binding partners lysis buffer DNA ratio p y Section 5 1 7 Sections 5 13 5 1 7 Section 5 1 7 Sections 5 1 7 www perkinelmer com alphatechnology 24 25 Cell based Assays 5 1 1 Selection and orientation of protein tags Documented cases showing successful use in conjunction with AlphaLISA include FLAG GST His and E Tag see Section 5 4 The use of a protein tag can be especially convenient when working with Alpha technology since the Table 6 Alpha reagents for Tag detection LEL AlphaScreen kits anti GST 6760603 Ni chelate 6760619 anti His anti c myc 6760611 anti FLAG 6760613 anti DIG 6760604 anti HA 6760612 anti FITC 6760605 anti V5 anti GFP anti Maltose Binding Protein MBP Strep Tactin expression of each protein can be confirmed before proceeding to the actual interaction assay see 5 1 6 PerkinElmer offers standalone detection reagents AlphaLISA Acceptor beads and Alpha Donor beads or complete AlphaScreen detection kits for these tags Table 6 AlphaLISA Acceptor beads Alpha Donor beads AL110 AL108 AS101 AL128 AL111 AL112 AS103 AL113 AL127 AL129 AL133 AL134 AL136 AS106 Please visit www perkinelmer com for additional product information an
33. ation Add 10 uL Acceptor beads and 10 uL Donor beads to each well final conc 20 ug mL each Second incubation Incubate 60 min at room temperature in dark place in a drawer with a TopSeal A adhesive seal or lid to prevent evaporation Read on an Alpha compatible reader EnVision Multilabel Plate Reader or EnSpire Multimode Plate Reader www perkinelmer com alphatechnology 12 Assay Development e This is a cross titration matrix For example well 5C will contain 3 nM Protein X and 30 nM Protein Y e The O nM wells are an important control and should not be omitted These are your background samples These wells will include both Donor and Acceptor beads 1 2 3 4 5 6 7 8 9 10 11 12 300nM 100nM 30nM 10 nM 3 nM 1 nM 0 3 nM 0 nM Empty Empty Empty Empty Protein X Protein X Protein X Protein X Protein X Protein X Protein X Protein X A 300 nM Protein Y B 100 nM Protein Y C 30 nM Protein Y D 10 nM Protein Y E 3 nM Protein Y F 1 nM Protein Y G 0 3 nM Protein Y H 0 nM Protein Y Figure 7 96 well plate map for cross titration experiment Notes e f your protein protein interaction is very strong K in pM range you may want to shift the range of tested concentrations lower If one of your proteins can bind more than one bead for example a protein that has two or more biotins can potentially bridge two streptavidin coated Donor beads you should add that particular associati
34. d available sizes As seen in the products table above some anti tag antibodies are available coupled to Donor beads allowing further assay flexibility for the user Note that the glutathione GSH detection system is not listed here and is not recommended for cell based applications as lysates contain high concentrations of free glutathione Cell based Assays The orientation of the selected tags on the recombinant proteins N or C terminal should be in accordance with current knowledge regarding the interaction of interest such that any possible obstruction toward the interacting regions is minimized When there is no available information on this the two proteins should be produced with the chosen tag in both the N and C terminal regions for a total of four different products The different tagging combinations can then be transfected in parallel to find the most optimal one s Section 5 1 6 will explain how to evaluate and compare the different tag orientations in terms of protein expression 5 1 2 Generation of expression vector The protein genes containing the chosen tags should be cloned into appropriate expression vectors 5 1 3 Transient cell transfection Many different technologies are available to transtect the expression vectors into cells Calcium Phosphate Electro poration Ballistic Particles DEAE Dextran Cationic Matrix and Lipofection The lipofection protocol is a relatively simple method that has been
35. d signal to background for detection of weak interactions Dissociation constants K for weak interactions can also be determined by using a competition assay rather than a satura tion assay See Section 4 for information on K determinations Antibody affinity If your assay is being designed using an antibody to associate a protein to a bead you may want to consider the use of weaker affinity antibodies when studying weak interactions K gt 100 nM If you are able to choose between antibodies with weaker or stronger affinities for a given protein you may find that more target protein can be added before saturating the beads when using a weaker affinity antibody coated bead If studying a weaker protein protein inter action it is desirable to use more protein in the assay to push the protein protein binding equilibrium to more protein protein complex By using a lower affinity anti body more protein can be added to the assay before saturating the bead However if the protein protein interaction being studied is strong you will likely generate a higher signal using a higher affinity antibody In this case less protein would be dissoci ated from the antibody at equilibrium Sample type The type of sample may also influence decisions regarding bead selection For example glutathione GSH beads and nickel chelate Ni beads may perform well in a biochemical assay but may not work well in a cell based assay This is
36. e assay conditions should also be optimized in terms of their effect on the actual protein protein interaction being studied Buffer salts Buffers used in biological assays will usually include a variety of salts to generate ionic strength and to satisfy the specific requirements of an assay Component such as an enzyme or protein Aloha assays are highly tolerant of the presence of a wide variety of salts and can tolerate the following ions up to 300 mM in solution Li Na Borate K Acetate Cs Bicarbonate Mg Carbonate Ca Phosphate monobasic and dibasic NH Sulfate F Pyrophosphate Cl Tartrate Br www perkinelmer com alphatechnology 8 Assay Design Heavy metals have to be considered carefully Alpha assays have shown sensitivity to heavy metals in solution most likely because those ions can react with singlet oxygen to Table 3 Interference levels in AlphaScreen TruPoint Assay form insoluble oxides Table 3 describes the effects of commonly used heavy metals on Alpha assay performance Recommended level Interference level IC Cobalt Co 0 7 mM or less 3 6 mM Iron ll Fe 2 mM or less 0 95 mM Iron 111 Fe 2 mM or less 9 mM Manganese Mn 7 mM or less 37 mM Nickel Ni 0 5 mM or less 2 8 mM Zine Zn 0 12 mM or less 2 69 mM Note Due to their steep inhibition curves a concentration of five times 5X less than the IC can be considered as having no effect on the assay
37. e beads competition The K value can then be calculated from the IC value based on a method introduced by Cheng and Prusoff Figure 18 Determining K in a Biochemical Assay Figure 18 Cheng and Prusoff Equation Binding AlphaScreen Definitions T target GPCR Protein X captured onto L tracer radioligand Acceptor beads K Protein Y captured d L T onto Donor beads 10 1 Rule of thumb T lt lt L lt lt KJ 0 1 nM 1 nM 10 nM 2 Cheng amp Prusoff e 1 ae Es K d L T Combining 1 amp 2 e K IC 1 1 7 K d L T 50 Therefore an IC determination from a carefully crafted competition assay should approximate the K The information above is written from the point of view of a optimized assay These protein concentrations should receptor radioligand binding assay In an Alpha protein protein be chosen from the first cross titration experiment interaction assay the target becomes one of the tagged see Section 3 1 keeping in mind that it is not neces proteins being caught to the bead and the tracer sary to pick the concentrations that give the highest becomes the second tagged protein binding to the other signal to background As long as the assay window is bead If the K is significantly higher than the concentration acceptable lower concentrations may be chosen of either protein the value for LI K in the Cheng amp Prusoff 2 The concentrations of each tagged pro
38. e performed on each of the double transfected cell lysates The assay should be set up to detect the interaction of the two overexpressed proteins under study Serial dilution of the lysate should correlate with a decreasing signal in the assay Ideally a negative control should also be performed using lysates of non transfected cells or cells transfected with a non related target cDNA Figure 24 shows the results of a serial dilution of lysate from a cell transfected with one ratio of Protein 1 FLAG Protein 2 GST DNA The Alpha assay employed a biotinylated anti GST antibody and anti FLAG conjugated Acceptor beads In this example the data confirmed that the amount of signal correlated with the amount of lysate added The assay was performed in a 384 well OptiPlate with a 25 uL final assay volume using the following protocol 1 Add 10 uL of CHO lysates prepared in lysis buffer 2 Add 5 uL Anti FLAG Acceptor beads 20 ug mL final diluted in assay buffer 3 Incubate 30 min at room temperature 4 Add 5 ul Biotinylated Anti GST antibody 1 nM final diluted in assay buffer 5 Incubate 60 min at room temperature Cell based Assays 6 Add 5 uL streptavidin Donor beads 20 ug mL final diluted in assay buffer 7 Incubate 60 min at room temperature 8 Read in Alpha compatible reader EnVision or EnSpire Multimode Plate Readers Competition protocol to validate protein protein interaction assay Where a recombinant for
39. e saturated with associating protein and when additional protein will not be able to associate with the bead If you have saturated a bead you may see a hooking effect Below the hook point both Donor and Acceptor beads become progressively saturated by the target molecule and the signal increases with increasing target concentration At the hook point either the Donor or the Acceptor component is saturated with the target molecule and a maximum signal is detected Above the hook point an excess of target molecules oversaturates the Donor or the Acceptor beads which inhibits their association and causes a progressive signal decrease Figures 3 and 4 The hook effect is a common phenomenon found when using any sandwich type assay for example ELISA assays Table 1 Alpha bead products and catalog numbers Toolbox Toolbox Alpha Donor beads Bead coating Streptavidin 6760002 Strep Tactin AS106 Anti GST Glutathione 6765300 Ni chelate AS101 Anti His Anti c myc Anti FLAG AS103 Anti DIG Anti HA Anti FITC Anti V5 Anti GFP Anti Maltose Binding Protein MBP Protein A AS102 6760137 Protein G Protein L Anti rabbit IgG AS105 Anti mouse IgG AS104 Anti human IgG Anti rat IgG Anti goat IgG Anti sheep IgG Anti mouse IgM Anti chicken IgY AlphaScreen Acceptor beads Assay Design Toolbox AlphaLISA Acceptor beads AL125 AL136 AL110 AL109 AL108 AL128 AL111 AL112 AL113 AL127 AL129 A
40. eads and Protein A AlphaLISA Acceptor beads were used in this assay Untagged EGF was titrated from 0 1 pM to 1 uM The expected K is 2 8 nM as determined by a radioligand binding assay Lax I et al 1988 Based on the data the calculated IC is 2 4 nM Essentially the same displacement assay used for assay validation see Section 3 4 will be used However it will be important to choose concentrations of the untagged protein that are centered around the expected Ky in order to derive an accurate IC Ideally a range of concentrations of untagged protein ranging from 0 01X to 100X the K would be used If the expected K is not known it may be neces sary to perform two experiments one using a very broad range of untagged protein concentrations Then once this data has been evaluated a second experiment can be set up using a narrower concentration of untagged protein Additionally it is advisable to test the assay with two or more different concentrations of tagged proteins that still adhere to the two criteria above to see if the IC changes significantly depending on the tagged protein concentra tion If the IC does vary dramatically between the two concentrations tested it is likely the lower concentration is more accurate the assay may need to be re run with still lower concentrations of tagged protein Figures 19 and 20 show two examples where competition binding experiments were used to determine K Figure 20 Co
41. ell type We therefore recommend testing several cell lysis buffers Some that have been successfully used with AlphaLISA are listed below A protease inhibitor cocktail should be added to all lysis buffers tested We recommend testing different lysis buffers to select the optimal one e 200 mM Tris HCl pH 7 5 1 Triton X 100 50 mM NaCl used for CHO cells see Waller et al e 100 mM Tris HCl pH 8 0 100 mM NaCl 0 5 NP40 used for BSR T7 5 cells see Rahman et al amp Mohamed et al e 50 mM Tris HCl pH 7 5 125 mM NaCl 5 glycerol 0 296 NPAO 1 5 mM MgCL 25 mM NaF 1 mM Na VO used for HEK 293T cells see Lavens et al e 50 mM Tris HCl pH 7 5 0 196 CHAPS used for CHO cells unpublished results from PerkinElmer www perkinelmer com alphatechnology 26 27 Cell based Assays We also recommend the following commercially available cell lysis buffers e AlphaLISA lysis buffer PerkinElmer Cat No ALOO3 proprietary formulation e Cell lysis buffer 10x Cell Signaling Technology Cat No 9803 1X Cell lysis buffer 20 mM Tris HCl pH 7 5 150 mM NaCl 1 mM Na EDTA 1 mM EGTA 1 Triton 2 5 mM sodium pyrophosphate 1 mM B glycerophosphate 1 mM Na VO 1 ug mL leupeptin e M PER Mammalian protein extraction reagent Thermo Scientific Cat No 78501 proprietary formulation e Pierce IP lysis buffer Thermo Scientific Cat No 87787 25 mM Tris HCl pH 7 4 150 mM NaCl 196 NP 40 1
42. enerate signal AlphaScreen Acceptor beads use rubrene as the final fluorophore emitting light between 520 and 620 nm AlphaLISA Acceptor beads use a Europium chelate as the final fluorophore emitting light in a narrower peak at 615 nm Figure 2 This makes the AlphaLISA Acceptor bead less prone to interference from buffer components or other chemicals that absorb light between 520 and 600 nm though this typically isn t a concern for protein protein interaction assays whether biochemical or cell based AlphaScreen Emission lphaLISA Emission E i 2 j E LL T m di E 500 520 540 560 580 600 620 630 650 680 700 nam Figure 2 Emission spectra of AlphaScreen and AlphaLISA Acceptor beads The decision of whether to use an AlphaLISA bead or AlphaScreen bead will likely depend most on convenience and type of bead coating offered e AlphaLISA beads are offered as individual toolbox reagents Donor beads are offered separately from Acceptor beads allowing the ability to mix and match bead options e AlphaScreen beads are typically packaged as kits pair ing streptavidin Donor beads with AlphaScreen Acceptor beads that bind to a particular tag or label as indicated in the product name The AlphaScreen kits also include an appropriate biotinylated probe such as biotinylated 6X His biotinylated GST tag etc that can be used as a positive control to check that the beads are working correctly 2 1 2 Bead
43. fer Assay Development 3 Prepare 200 uL of a 4X working solution 1 2 UM of Protein Y in your assay buffer 4 Perform a serial dilution of the 1 2 uM stock of Protein Y in Eppendorf tubes as follows Final M Intermediate M 4X Vol of dilution Assay Buffer 3x 107 O 200 uL of 1 2 uM Protein Y B 1X10 Ax 10 66 uL of Tube A 132 uL C 3X MOF 12 X ice 66 uL of Tube B 154 uL D TXIO 4X 10 66 uL of Tube C 132 uL E 3X 107 12 X 107 66 uL of Tube D 154 uL F 1X 107 AL O 66 uL of Tube E 132 uL G BG One 2S 66 uL of Tube F 154 uL H 0 0 300 uL of buffer 5 During first incubation refer to protocol below Prepare a 4X working solution 80 ug mL of Acceptor beads 16 uL Acceptor beads 5 mg mL 984 uL assay buffer 6 During first incubation refer to protocol below Prepare 4X working solution 80 ug mL of Donor beads keeping protected from light 16 uL Donor beads 5 mg mL 984 uL assay buffer B Assay protocol for a 96 well Y AreaPlate Total assay volume of 40 pL Refer to the plate map Figure 7 You can use a multi channel repeat pipettor to quickly dispense reagents into the plate Add 10 uL Protein X to each well referring to the plate map on next page final conc O nM 300 nM Add 10 uL Protein Y to each well referring to the plate map on next page final conc O nM 300 nM First incubation Incubate 60 min at desired temperature with a TopSeal A adhesive seal or lid to prevent evapor
44. his kit contains streptavidin coated Donor beads and biotinylated AlphaScreen Acceptor beads which interact in the absence of any binding partners TruHits beads are commonly used as a control when evaluating potential interference from buffer components or from compounds being tested in the assay Other bead types may exhibit different sensitivities to various buffer components for example a nickel chelate bead is more sensitive to EDTA than a streptavidin bead Additionally while some buffer aspects may not affect the Alpha signal itself they may affect the conformation or interaction of the proteins being studied or the associations of these proteins with the beads 2 2 1 General concepts Buffer choice can be very important Choose pH buffering capacity and salt concentration that will facilitate the desired interactions between the components of your assay If metal co factors are needed for correct conformational integrity of your proteins it is best to titrate these components appro priately as certain metal ions can interfere with Alpha assays If excessive non specific binding is observed a variety of different detergents may be used such as Tween 20 0 01 0 1 Triton X 100 0 01 0 1 or CHAPS 0 1 or less For most Alpha applications a BSA concentration of 0 1 w v is sufficient to minimize non specific interactions Some assays may require slightly higher concentrations of BSA Try to avoid azide as a preservative
45. in Alpha assays www perkinelmer com alphatechnology 10 Assay Development 3 Assay development biochemical protein protein interaction assays The information in this section presents a possible series of experiments to perform to optimize your Alpha protein protein interaction assay Before you begin e Alpha assays require a special reader capable of measur ing an Alpha assay Only PerkinElmer multimode detec tion plate readers have been validated to read Alpha technology Alpha technology has not been optimized by PerkinElmer for use with other systems e We recommend preparing only what you need for the day s experiments Do not store working dilutions of beads for more than one day e The Donor beads used in Alpha assays are somewhat light sensitive We recommend working under subdued lighting conditions when working with the beads less than 100 Lux the level of light produced on an overcast day For example you can turn half of the laboratory lights off and work at a bench away from windows and where the overhead light is not on Incubate the plate in the dark for example placing the covered or sealed plate in a drawer e The Alpha signal is temperature dependent If you will be performing incubations at 37 C or other temperatures we recommend that you equilibrate the plate back to room temperature before reading to ensure signal unifor mity across the plate 3 1 The first experiment protein cross tit
46. in interaction may be determined with Alpha assays using one of two approaches saturation curves section 4 1 or competition assays section 4 2 Of these competition assays are applicable to a wider range of assay conditions 4 1 Saturation curves for determination of K d In limited situations saturation curve assays can be performed to determine the affinity constant K for the protein protein interaction in Alpha format In a saturation assay one binding protein is titrated across a range of concentrations to generate a saturation curve The K can be derived from the data as the protein concentration at which half maximal signal is reached Saturation curves can be used to determine K only if the assay meets the following criteria 1 The K for the protein protein interaction is far enough below the binding capacity of each bead so that all protein concentrations used to derive the K usually up to 5X K fall below the bead binding capacities This typically means that the K needs to be in the sub nanomolar range though some beads have a binding capacity up to 300 nM refer to Table 2 2 There is an excess of the binding partner that will be varied in comparison to the concentration of the fixed binding partner For example if you are vary ing the concentration of Protein Y in the saturation curve Protein Y should be in at least 10X excess molar concentration compared to Protein X Other wise there is a ligand
47. iol 332 1131 1141 2003 Lavens D et al Definition of the interacting interfaces of Apobec3G and HIV 1 Vif using MAPPIT mutagenesis analysis Nucleic Acids Res 39 1902 12 2009 Lax et al Chicken epidermal growth factor EGF receptor cDNA cloning expression in mouse cells and differential binding of EGF and transforming growth fac tor alpha Mol Cell Biol 8 1970 1978 1988 10 11 Lazar G A et al Engineered antibody Fc variants with enhanced effector function Proc Natl Acad Sci U S A 103 4005 10 2006 Mohamed M R et al Proteomic screening of variola virus reveals a unique NF kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses Proc Natl Acad Sci U S A 106 9045 9050 2009 Rahman M M Mohamed M R Kim M Smallwood S 8 McFadden G Co regulation of NF kappaB and inflammasome mediated inflammatory responses by myxoma virus pyrin domain containing protein m013 PLoS Pathog 5 e1000635 2009 Waller H Chatterji U Gallay P Parkinson T amp Targett Adams P The use of AlphaLISA technology to detect interaction between hepatitis C virus encoded NS5A and cyclophilin A J Virol Methods 165 202 210 2010 Werden S J amp McFadden G Pharmacological manipu lation of the akt signaling pathway regulates myxoma virus replication and tropism in human cancer cells J Virol 84 3287 3302 2010 www perkinelmer com alphatechnology 38 PerkinElmer I
48. ion 5 2 5 The detection of an interaction between endogenously expressed proteins must rely on the use of specific antibod les since the proteins will not have expression tags As for the tagged overexpressed proteins discussed in section 5 1 it is crucial to gather good evidence that the two endog enous proteins to be studied for interaction are indeed expressed in the cellular model chosen In the case of endogenous non tagged proteins the most popular technique for achieving this is the Western blot One can also use an antibody based Alpha assay to detect each protein individually In this instance a variety of antibodies should be tested in order to maximize the chance of finding at least one robust pair of antibodies for the assay When data from ELISA experiments are available one should first select the same pair of antibodies to try in an Alpha assay If this is not successful other antibody pairs can then be investigated 5 2 1 Antibody selection The selection of antibodies should be made based on the available literature for the interaction The chosen antibodies should bind to an epitope that is as distant as possible from the interacting domain s of the proteins to avoid steric hindrance When the map of the interaction is unknown we suggest selecting and testing multiple antibodies that target different regions of the protein Combinations of these antibodies can then be tested in a matrix experiment to find the
49. ity will be influenced by size of the protein associating with the bead as well as affinity of the protein or antibody for the bead Protein A interacts strongly with particular subclasses of antibodies including human lgG1 IgG2 and IgG4 mouse IgG2A and IgG2B and rabbit human and mouse total IgG Protein A also has weaker affinity for other antibody subclasses The bead capacity for these products refers to the capacity for antibody The amount of protein you will then be able to bind to the antibody on the bead will be dependent on the affinity of your antibody for your protein and the size of your protein and may range anywhere from 1 pM to 1 uM Protein G binds to all subclasses of human IgG and mouse IgG In addition it binds to rat goat sheep guinea pig rabbit cow pig and horse antibodies gt Protein L binds efficiently total human IgG IgM IgA IgE IgD mouse IgG and rat IgG It binds only poorly mouse IgM and rabbit IgM and it does not bind human IgG lambda light chain rabbit sheep goat and bovine IgG and rat IgM Assay Design 3 g Alpha Signal counts E a 412 41 10 3 8 7 6 5 4 3 Log Analyte g mL 329 339 LOW ANALYTE CONCENTRATION Some binding sides aecupied OPTIMAL AMALYTE CONCENTRATION Optimal amp banding siles occupied Maximam signal production Optimal bead association Signal production above background Limited bead association Streptavidin coated Alpha Donor Boad
50. lated with the amount of lysate added to the assay 5 2 5 Alpha interaction assay for endogenous proteins Matrix experiments The initial search for a positive signal in an Alpha assay demonstrating a protein protein interaction event will involve exploring two important parameters lysis buffer and antibody selection The first experiments should consist in matrix assays where all the combinations of the various lysis buffers and antibody pairs will be tested in parallel Figure 26 Please see section 5 1 7 for help in selection of the assay buffer in which the protein protein interaction will be performed prior to cell lysis www perkinelmer com alphatechnology 32 Cell based Assays For each antibody pair tested we recommend including a negative control for which no lysate is added lysis buffer only Alternatively if the interaction is known to be modulated by a cell treatment treated versus untreated cell lysates might serve as positive and negative controls This should help discard any potential cross reactivity or non specific signal that could mistakenly be thought to originate from the interaction occurring between proteins Lysis Buffers mE Pairs Figure 26 Example of a matrix assay for selection of a lysis buffer and antibody pairs In this 384 well microplate setup four lysis buffers and eight antibody pairs are tested in triplicate Confir
51. lence 20 000 corresponding to the undiluted lysates Untransfected CHO WT lysates were included as a negative control Results indicated that expression levels were distinctly higher when the GST tag was located at the C terminal end of Protein 2 www perkinelmer com alphatechnology 28 29 Cell based Assays Additional ways to assess protein expression The experimental examples given here can help in confirming the expression of the tagged proteins However this method ology does not constitute a definitive proof that the cell expresses a fully functional tagged protein For example the protein may be truncated or cleaved and the tag could not be attached to the protein We therefore suggest performing a Western blot detection to verify that the expressed tagged proteins display the expected molecular weights If an antibody is available to the tagged protein one could use an Alpha assay where the tagged protein is detected using both the tag and a specific antibody in a capture assay format Compared to the tag based detection strategy described in section 5 1 6 this type of assay can provide more compelling proof for the functional expression of the tagged proteins since the antibody epitope and the tag need to be present on the same protein to generate an Alpha signal Based on the results obtained in this section the best constructs and the optimal lysis buffer should be selected for use in subsequent double transfection and p
52. luted in 1 10 in assay buffer separated following centrifugation for 10 min at 13 000 g at 4 C Rahman et al 100 mM Tris pH 8 0 100 mM GST and His Forty eight hours post transfection cells PBS 0 1 BSA Cells seeded in 24 well plates PLoS Pathogens 2009 Lavens et al Nucleic Acids Research 2009 Mohamed et al PNAS 2009 Werden et al Journal of Virology 2010 Waller et al Journal of Virological Method 2010 Internal data presented in this guidebook Etag and FLAG His and GST His FLAG and HA FLAG GST His and HA His and HA for transfected cells FLAG and GST NaCl 0 5 NP 40 containing Roche complete protease inhibitor 50 mM Tris HCl pH 7 5 125 mM NaCl 5 glycerol 0 2 NP40 1 5 mM MgCl 25 mM NaF 1 mM Na VO Complete protease inhibitor without EDTA cocktail Lysates were cleared by centrifugation 100 mM Tris pH 8 0 100 mM NaCl 0 596 NP 40 containing Roche complete protease inhibitor Not disclosed Following a PBS wash 200 mM Tris HCl pH 7 5 1 Triton X 100 50 mM NaCl Bulk produced at 4M ml in 50 mM Tris HCl pH 7 4 0 1 CHAPS protease inhibitor cocktail note CHAPS was documented as a detergent that preserves the studied interaction were collected in 500 mL PBS pelleted and then lysed by suspending in 20 mL lysis buffer The cellular extracts were then separated following centrifugation for 5 min at 13 000 rpm Forty eight hours post
53. m of one of the target proteins is available this can be added to the double transtected cell lysate in increasing concentrations Figure 25 A reduction in signal with increasing concentrations of this competing protein suggests that the assay is detecting the desired protein protein interaction One essential requirement for such a competition experiment is that the recombinant protein must not contain either of the two tags used in the detection assay FLAG and GST in this example The assay shown here was performed as follows in a 384 well Opti Plate using a 25 uL final assay volume 10 000 7 500 5 000 2 500 0 eS SG A e Number of cells well lysates Figure 24 Alpha assay detection of tagged Protein 1 Protein 2 interaction illus trating that the signal correlates with varying amounts of protein protein complex Lysate dilutions prepared from cells transfected with Protein 1 FLAG Protein 2 GST DNA were tested in an Alpha assay AlphaScreen Anti FLAG Acceptor beads and streptavidin Donor beads were used at 20 ug mL final and biotinylated anti GST was used at 0 3 nM final 1 Add 5 uL Anti FLAG Acceptor Beads 20 ug mL final diluted in assay buffer 2 Add 5 uL recombinant Protein 2 diluted in assay buffer 3 Add 5 uL CHO lysates prepared at 4M cells mL 4 Incubate 60 min at room temperature 5 Add 5 uL of biotin Anti GST 1 nM final diluted in assay buffer 6 Incubate 60 min at room temperature
54. mM EDTA 5 glycerol Preparation of lysis buffer 1 Dilute the lysis buffer to 1X if needed prior to use 2 Add a standard protease inhibitor cocktail Sigma Cat No P2714 or Roche Cat No 05 892 791 001 to the lysis buffers prior to use Reconstitute according to the manufacturer s instructions Other cocktails can be used as well Cell lysis procedure 1 Treat the cells if needed Section 5 1 4 2 Detach the cells if required 3 Determine the cell density of the culture Harvest the cells by centrifugation 4 Discard the supernatant and resuspend the cells in 1X Lysis buffer The optimal number of cells must be determined experimentally However 4 x 109 cells mL are recommended as a starting point Add 5 uL of 1X Lysis buffer to 20 000 cells 5 Incubate for about 5 10 minutes at room temperature on a rocker to maximize the lysis 6 Centrifuge for 5 minutes at high speed 13 000 RPM in a benchtop centrifuge to clarify the lysate Discard the pellet 7 Prepare aliquots and freeze at 80 C 5 1 6 Protein expression assessment Once the bulk cell lysates are prepared as a preliminary step it is important to evaluate the relative levels of expression of each protein in the cells We cannot stress enough the importance of gathering solid evidence for expression of the respective proteins before proceeding to the actual detection of the interaction Although this does not guarantee success in measuring a protein
55. mation that a positive signal is a valid protein protein interaction Refer to section 5 1 7 for examples of experiments that can confirm the validity of your protein protein interaction Alpha assay 5 3 Studying protein protein interactions on non tagged recombinant proteins Cell transfections using Preparation of Protein expression assessment various DNA amounts cell lysates using by Western blot and or Alpha each target expressed various lysis assay identification of individually buffers optimal lysis buffer Titration of various cell lysates in Alpha assay for identification of optimal DNA ratio Preparation of cell lysates using optimal lysis buffer Cell transfections at variable DNA ratios coexpression of binding partners Positive signal confirmation in Alpha assay 33 Cell based Assays The approach taken here is a combination of the fundamen tals covered in Sections 5 1 and 5 2 It relies on specific antibodies directed against the recombinant proteins and for that reason these assays are more challenging than those involving tagged recombinant proteins On the positive side they are in general easier to develop than the assays for endogenous proteins due to the higher expression levels of recombinant proteins See sections 5 1 2 through 5 1 5 for information on prepar ing the cell lysates for analysis s for other applications lysates should be prepared and tested from
56. me optimization up to overnight Start up Guide Cell based Assays Lysis buffer selection The measurement of cell based protein protein interactions using Alpha requires the production of cell lysates We recommend testing several cell lysis buffers A protease inhibitor cocktail such as Sigma Aldrich Cat No P2714 or Roche Cat No 05 892 791 001 should be added to all lysis buffers tested The optimal number of cells must be determined experimentally For the protocols below we recommend preparing a lysate equivalent to 2 x 10 cells mL lysis buffer Some recommended lysis buffers e AlphaLISA lysis buffer PerkinElmer Cat No ALOO3 e Cell lysis buffer 10x Cell Signaling Technology Cat No 9803 e M PER Mammalian protein extraction reagent Thermo Scientific Cat No 78501 e Pierce IP lysis buffer Thermo Scientific Cat No 87787 Protein expression assessment Once the bulk cell lysates are prepared it is important to evaluate the relative levels of expression of each protein in the cells For tagged proteins this can be done with a competition type assay using the tagged probe included in an appropriate AlphaScreen kit For endogenous proteins this For tagged protein protein interaction events the first experiment will involve evaluating lysis buffer and amount of lysate Cell lysates should be diluted in assay buffer to reduce lysis buffer interference can be done using
57. minute incubation 2 Protein A AlphaLISA Acceptor bead addition 60 minute incubation 3 Streptavidin Donor bead addition 30 minute incubation followed by reading the plate The streptavidin Donor beads were added last after the second incubation to prevent the multi biotinylated EGF from bridging two streptavidin Donor beads before other interactions could occur www perkinelmer com alphatechnology 20 21 Determining K in a Biochemical Assay Saturation curves for Kd determination 300 000 e 1nMEGFR Fc 0 3 nM EGFR Fc 200 000 A 0 1 nM EGFR Fc Alpha signal 4 6 10 Biotin EGF nM Ll 1nM EGFR Fc 0 3nM EGFR Fc 0 1nM EGFR Fc Bmax 386709 202583 78954 Kd 4 642 3 902 3 312 Figure 16 Saturation curve used to determine K of the interaction between biotinylated EGF and EGFR Fc in Alpha format The expected K for this interaction is 2 8 nM This approach is generally suitable for low sub nanomolar K values Example data where K cannot be determined by a saturation curve In Figure 17 the expected K of the interaction between HDM2 and p53 peptide is 0 3 uM This is well above the expected binding capacity of the beads used in the Alpha assay which is an indication that the saturation method should not be used to determine K instead a competition binding assay should be used Based on these data the K would be incorrectly derived to be 2 nM 10 nM biotinylat ed p53 peptide was used in the
58. mpetition binding assay to determine K for a biotinylated p53 GST HDM2 protein protein interaction Streptavidin Donor beads and anti GST AlphaScreen Acceptor beads were used in this assay A few concentrations of tagged protein GST HDM2 were tested and the IC values derived were fairly consistent Untagged p53 was titrated from 30 nM to 100 uM Concentration of biotinylated p53 used in the assay was 10 nM The expected K for this interaction is 0 3 uM Dawson R et a 2003 Compare to Figure 17 where data derived from a saturation curve erroneously gave a K of 2 nM Why can a signal be obtained even when working at tagged protein concentrations that are well below the K Because Alpha beads have avidity multiple binding sites per bead the microenvironment of the bead artificially increases the concentration of tagged protein in the vicinity of the bead An excellent example of the use of competition binding curves to determine affinity in an Alpha assay format is in a paper by Lazar et al 2006 The IC of the interaction between three Fc variant antibodies and Fcgamma receptor was determined to be 380 nM 60 3 nM and 7 94 nM respectively These IC s were in close agreement with SPR surface plasmon resonance derived K values 252 nM 30 nM and 2 nM respectively Cell based Assays 5 Cell based assays While performing biochemical assays is the most straightfor e Alpha assays involving endogenously expressed p
59. nc 940 Winter Street Waltham MA 02451 USA P 800 762 4000 or 4 1 203 925 4602 www perkinelmer com Perki n For a complete listing of our global offices visit www perkinelmer com ContactUs Copyright 2011 PerkinElmer Inc All rights reserved PerkinElmer is a registered trademark of PerkinElmer Inc All other trademarks are the property of their respective owners 009625 01 May 2011
60. ng bead for e If you are using weak affinity beads and your protein example the streptavidin Donor beads last to allow protein interaction is weak K gt 100 nM you may want other interactions to occur first Refer to Order of to include higher concentrations on this plate addition Section 3 3 Assay Development In the protocol for the following data Figures 8 and 9 a 3 step assay was performed biotinylated EGF and EGFR Fc added first followed by a 60 minute incubation Protein A AlphaLISA Acceptor beads added second followed by a 60 minute incubation and streptavidin Donor beads added last with a final 30 minute incubation before reading the plate The streptavidin Donor beads were added last after the second incubation to prevent the multi biotinylated EGF Figure 8 Cross titration data for EGFR Fc binding to biotinylated EGF using strep tavidin Donor beads and Protein A AlphaLISA Acceptor beads The expected K for this interaction is 2 8 nM Lax I et al 1988 Cross titration biotin EGF binding to EGFR Fc 500 000 4 Hook point 100 nM EGFR Fc 30 nM EGFR Fc 10 nM EGFR Fc 3nM EGFR Fc 1nM EGFR Fc 0 3 nM EGFR Fc 0 1 nM EGFR Fc 0nM EGFR Fc 400 000 300 000 200 000 E z E 100 000 4 0 lt gt B log biotin EGF M Figure 9 Titration curve in GraphPad Prism of the same data presented in Figure 8 from bridging two streptavidin Donor beads before other interactions could
61. oated bead used at 20 ug mL usually saturates at around 30 nM biotinylated peptide but will saturate at around 2 3 nM biotinylated antibody e Affinity of the bead for the associating reagent for a GST tagged protein one can choose either anti GST antibody coated beads or glutathione beads However the affinity of glutathione for GST is weaker compared to the affinity of anti GST antibody for GST This means that one would be able to add higher concentrations of GST tagged protein to a glutathione bead before satu rating hooking because for a given concentration of the protein more GST tagged protein is not associated with the glutathione bead You may find that the hook point is reached on anti GST beads at around 20 nM GST tagged protein but at around 200 nM GST tagged protein on glutathione beads For weak interactions use of a weaker affinity bead may be desirable Flgure 6 For strong protein protein interactions the protein concen tration may not need to be titrated to as high a level and a stronger signal may be obtained with a higher affinity bead because less protein is dissociated from the bead A note about bead capacity Weak protein protein interactions even in the high uM or low mM affinity range can be studied using Alpha even though the maximum bead capacities tend to fall in the nM range The high signal produced by Alpha assays usually means that you can choose nM concentrations of proteins and still get a goo
62. oking effect A cross titration was performed to characterize the interaction between biotinyl ated mouse EGF and Fc fusion human EGFR receptor Streptavidin Donor beads and AlphaLISA Protein A Acceptor beads were used to capture the proteins and generate the assay signal The expected K for this interaction was 2 8 nM Lax etal 1988 A hook point is reached at 10 nM biotinylated EGF and 3 nM EGFR Fc Saturation binding HDM2 binding to p53 400 000 300 000 200 000 e Total Binding A Non Specific Binding Alpha signal 100 000 4 6 8 GST HDM2 nM Figure 5 Low affinity protein protein interaction on low capacity beads illustrating how the overlapping effects of hooking and increasing protein protein interaction can resemble a saturation curve The interaction studied is between biotinylated p53 and GST tagged hDM2 Streptavidin Donor beads and Anti GST AlphaScreen Acceptor beads were used to capture the ligands and generate the assay signal The expected K for this interaction was 0 3 pM Dawson R et a 2003 but deriving a K value from the half maximal signal would give an incorrect value of 2 nM See Section 4 for K calculation using Alpha assays www perkinelmer com alphatechnology 6 Assay Design Bead capacities are influenced by e Size of the protein associating with the bead more small peptides will fit on the surface of a bead compared to large antibodies For example a streptavidin c
63. optimal pair A practical search engine for commercially available antibodies can be found in the Biocompare site www biocompare com When available recombinant versions of the studied proteins could prove helpful in selecting antibodies and possibly in confirming the specificity of the putative protein protein interaction When testing different antibody pairs in an Alpha assay the assay can be relatively quickly developed using a recombinant protein as the target Once the protein protein assay format has been established the recombinant protein can be used to show assay specificity by competing with the interaction of the endogenous proteins being studied See section 5 1 7 for an example of this approach using overexpressed proteins 5 2 2 Antibody labeling All of the selected antibodies should be prepared for use in both of the possible orientations in the Alpha assay In other words an aliquot of each antibody should be biotinylated for capture on streptavidin Donor beads and a separate aliquot should be directly coupled to Acceptor beads Protocols are supplied in the AlphaLISA Assay Development Guide For each antibody pair the Alpha assay should be tested in both configurations i e with Antibody 1 Acceptor beads and biotinylated Antibody 2 and with Antibody 2 Acceptor beads and biotinylated Antibody 1 For some antibodies where the formulation prevents bead coupling or biotinylation secondary antibodies can be u
64. other ultimately producing a luminescent signal Alpha assays require two bead types Donor beads and Acceptor beads Each bead type contains a different proprietary mixture of chemicals which are key elements of the Alpha technology Donor beads contain a photosensitizer phthalocyanine which converts ambient oxygen to an excited and reactive form of O singlet oxygen upon illumination at 680 nm Within its 4 usec half life singlet oxygen can diffuse approximately 200 nm in solution If an Acceptor bead is within that distance energy is transferred from the singlet oxygen to thioxene derivatives within the Acceptor bead resulting in light production at 520 620 nm AlphaScreen or at 615 nm AlphaLISA If the Donor bead is not in proximity of an Acceptor bead the singlet oxygen falls to ground state and no signal is produced Figure 1 In an Alpha protein protein interaction assay one protein is captured on the Donor beads and the other protein is captured on the Acceptor beads When the two proteins interact the Donor bead is brought into proximity of the Acceptor bead and excitation of the Donor bead will result in signal generation dependent on the presence of an Interaction GST tagged HDM2 Biotinylated P53 Streptavidin coated Anti GST conjugated Alpha Donor Bead AlphaLISA Acceptor Bead Figure 1 Illustration of an Alpha protein protein interaction assay using streptavi din coated Donor beads AlphaLISA anti
65. protein cross titration experiment This interaction is between His tagged MEK1 and GST tagged ERK2 with capture and detection using glutathione Donor beads and nickel chelate AlphaLISA Acceptor beads The expected K for this interaction is 29 uM Bardwell A J et al 2001 A hook point is reached at 100 nM GST ERK2 and between 100 nM and 300 nM His MEK1 www perkinelmer com alphatechnology 14 Assay Dev 1nM 0 1nM elopment 11 12 7 9 10 0 nM 1 uM 100nM 10nM 1 nM 0 1 nM 0 nM Protein X Protein X Protein X Protein X Protein X Protein X Protein X Protein X Protein X Protein Y Protein Y Protein Y Protein Y F 1 nM Protein Y G 0 3 nM Protein Y Protein Y m H 0 nM Protein Y MN m EE Figure 11 Suggested 96 well plate map for cross titration experiments that involve the use of an antibody to capture Protein X to beads 3 3 Order of addition Order of addition can influence the signal generated to a large extent The optimal order in which assay components interact should always be determined empirically Keep in mind that some binding partners may interfere with the association of other binding partners if allowed to interact in the wrong order The protocol presented in Section 3 1 is a two step protocol the two proteins are added first and incubated and then the two beads are added in a second addition before the final incubation It is possible to take the protocol and split this into three steps
66. ration The first experiment is a protein cross titration In this experiment you will be keeping the concentrations of beads constant 20 ug mL final concentration of each bead and varying only the concentration of each protein The plate map for this assay will be designed to test multiple possible combinations of each protein s concentration in a matrix The assay is performed in singlicate one well per condition Note This protocol assumes you are performing an assay that uses two proteins that bind directly to the Donor and Acceptor beads without the use of an antibody If you also have an antibody in your assay please refer to Section 3 2 Cross titration when you are using antibodies to capture proteins to beads A Preparation of reagents 1 Prepare 200 uL of a AX working solution 1 2 uM of Protein X in your assay buffer If you are not sure what assay buffer to use try using 1X PBS 0 5 96 BSA The BSA is important to prevent non specific interactions 2 Perform a serial dilution of the 1 2 uM stock in Eppendorf tubes as follows Tube Finall M Intermediate M 4X Vol of dilution Assay buffer 1 3 x 107 278 HOR 2 1X107 4 x 107 3 SIX qe 12 X 108 4 A MOS 4 X 10 5 SAO 12 X 10 6 1 X 10 4 X 10 7 A TOFU 12 X 101 8 0 0 200 uL of 1 2 uM Protein X 66 uL of Tube 1 132 uL 66 uL of Tube 2 154 uL 66 uL of Tube 3 132 uL 66 uL of Tube 4 154 uL 66 uL of Tube 5 132 uL 66 uL of Tube 6 154 uL 300 uL of buf
67. rotein protein interaction experiments 5 1 7 Alpha protein protein interaction assay When you are ready to perform the double transfection experiments for the actual detection of an interaction we suggest that various ratios of DNA be tested in transfections since a 1 1 stoichiometry in DNA amount will not necessarily result in comparable expression levels for the two proteins Various factors may affect the efficiency of transfection protein transcription and maturation Selection of assay buffer While there is no universal assay buffer that can be recom mended to suit all protein protein interaction studies the following two buffers were shown to be successful in some Cases e PBS 0 196 BSA ref Rahman et a Mohamed et al e 50 mM Tris HCl pH 7 4 150 mM NaCl 0 1 BSA PerkinElmer unpublished results In addition test any assay buffers that have been documented in the literature for use with the protein protein interaction of interest One general recommendation is to avoid the use of detergents in the assay buffer composition Some detergent will most likely be introduced to the assay by the sample The sample lysis buffer will be diluted 5 fold if 5 uL of sample is added in a 25 uL assay Excessively high levels of certain detergents could affect the protein protein interac tion and the Alpha technology as well Also it is important to verify that the buffer does not contain substances known to interfere with the
68. roteins ward approach to study protein protein interactions often the success of these assays will greatly depend on the the cellular environment can be essential to generate the endogenous levels of expression of each protein and the proper interaction Cell based assays to study these interac availability of specific antibodies directed against the two tions using Alpha assays can roughly be divided into three proteins see Section 5 2 care genes e Alpha assays involving non tagged recombinant proteins e Alpha assays involving tagged recombinant proteins transfected into cells while this approach relies on anti transfected into cells this approach is the preferred one body detection it presents an advantage over the second due to the high protein expression levels achieved in approach due to higher amounts of proteins expressed in transfected cells and the availability of good anti tag transfected cells see Section 5 3 antibodies see Section 5 1 5 1 Studying protein protein interactions using overexpressed tagged proteins Cell transfections using Alpha assay protein Selection of tags and various DNA amounts Preparation of expression assessment tag orientation each target expressed cell lysates using identification of best tag Section 5 1 1 individually various lysis buffers orientation and optimal Sections 5 1 2 5 1 3 Sections 5 1 4 5 1 5 lysis buffer Section 5 1 6 Cell transfections
69. s skewing the data A competition binding curve should be tested for K determination 2 K is calculated to be 5 10 nM when using streptavidin beads Because the streptavidin biotin interaction is a relatively tight interaction the binding capacity of these beads is fairly predictable If the calculated K is between 5 and 10 nM and streptavidin beads are being used it is likely the binding capacity of the beads has been exceeded A competition binding curve should be tested for determination of K Example data An excellent example of the use of saturation curves to determine K in an Alpha assay format is in a paper by Cassel et al 2010 In this work the K of the interaction between TAR DNA binding protein 43 TDP 43 and biotinylated single stranded TAR DNA and 6 TG repeats was determined to be 0 75 nM and 0 63 nM respectively Association and dissociation rates were also calculated and used to derive K values In both cases the K derived was consistent with previous reports using other methods In Figure 16 saturation curves were used to determine the K for the interaction between biotin murine EGF and Fc fusion human EGFR Streptavidin Donor beads and Protein A AlphaLISA Acceptor beads were used in the assay The expected K is 2 8 nM as determined by a radioligand binding assay Lax I et al 1988 In the protocol for this data a 3 step assay was performed as follows 1 biotinylated EGF and EGFR Fc addition 60
70. sed as an alternative capture method e g AlphaLISA Anti mouse IgG Acceptor beads so long as there is no problem of cross reactivity to the other antibody in the assay PerkinElmer offers stand alone detection reagents and complete detection kits along with Donor beads for these antibodies Table 7 Cell based Assays Table 7 Alpha beads for indirect antibody capture AlphaScreen kits AlphaScreen AlphaLlSA Alpha Donor beads Acceptor beads Acceptor beads Protein A 6760617 6760137 AL101 AS102 Protein G AL102 Protein L AL126 Anti human IgG AL103 Anti rabbit IgG 6760607 AL104 AS105 Anti mouse IgG 6 60606 ALTOS AS104 Anti rat IgG AL106 Anti goat IgG AL107 Anti sheep IgG AL132 Anti mouse IgM AL130 Anti chicken IgY AL131 Fc specific antibodies Please visit www perkinelmer com for additional product information and available sizes 5 2 3 Cell lysis Please consult section 5 1 5 for detailed information on cell lysis 5 2 4 Protein expression assessment Once the bulk cell lysates are prepared a preliminary step consists in evaluating the relative level of expression of each protein in the cells This can be done in an Alpha assay by capturing each individual protein with specific antibodies or by Western blot analysis Different lysis buffers may be tested at this stage As for tagged proteins section 5 1 6 it is important to verify that the assay signal is specific for the targeted protein and is corre
71. t ox A 26 5 1 3 Transient cell transfection lll ll s 26 DA ell IEEE 26 Sd GCIs x rcs x 524343945994495959924245 7 55 52342 259 9243u5 26 5 1 6 Protein expression assessment 2 lll lll ll ll Ss 2 5 1 7 Alpha protein protein interaction assay lle 29 5 2 Studying protein protein interactions using endogenous proteins lll 31 5 2 1 Antibody selection gt xx be who3 9 xS wc P SDE ss 31 SAA ATO TOS Eu s a urb E EROR RAE ss We d OR ROBORE AA XR Ode Ro EG 31 5 2 3Celllysis ol sess ss 32 5 2 4 Protein expression assessment 2 ooo a a a a a a 32 5 2 5 Alpha interaction assay for endogenous proteins a a a a a a 32 5 3 Studying protein protein interactions on non tagged recombinant proteins 2 33 5 4 Cell based references lll ls 34 Quick Start Guide to Alpha Protein Protein Interactions lol sls 35 Microplates for Alpha assays and recommended volumes llis 37 Scaling the assay volumeupordown oo sn 37 Referentes 38 Introduction 1 Introduction AlphaScreen and AlphaLISA are bead based assay tech nologies used to study biomolecular interactions in a microplate format The acronym Alpha stands for Ampli fied Luminescent Proximity Homogeneous Assay The assay does not require any washing steps Binding of proteins or other binding partners captured on the beads leads to an energy transfer from one bead to the
72. ted rabbit IgG with streptavidin bead Protein L Antibodies 1 nM antibody Biotinylated human IgG kappa with streptavidin beads Anti human IgG Fc portion of human IgG antibodies 3 nM antibody Biotinylated human IgG with streptavidin bead Anti rabbit IgG Fc portion of rabbit IgG antibodies 1 nM antibody Biotinylated rabbit IgG with streptavidin bead Anti mouse IgG Fc portion of mouse IgG antibodies 3 nM antibody Biotinylated mouse IgG with streptavidin bead Anti mouse IgM Mouse IgM immunoglobulins 0 3 nM antibody Biotin mouse IgM with streptavidin beads Anti rat IgG Fc portion of rat IgG antibodies 1 nM antibody Biotinylated rat IgG with streptavidin bead Anti goat IgG Fc portion of goat IgG antibodies 3 nM antibody Biotinylated goat IgG with streptavidin bead Anti sheep IgG Fc portion of sheep IgG antibodies 1 nM antibody Biotinylated sheep IgG with streptavidin beads Anti chicken IgY Fc fragment of chicken IgY immunoglobulins 0 3 nM antibody Biotinylated chicken IgY with streptavidin beads The numbers provided are examples given to compare relative bead capacities and are derived from probe titration experiments as shown in the QC data on the product tech data sheets www perkinelmer com coa The actual bead capacity for your bead system should be determined empirically for a given assay The bead capacities given in this table are influenced by partner bead and the size of the probe used in these assays The actual bead capac
73. tein used in the equation approaches zero The full equation then becomes assay need to be below the binding capacity of their K IC 140 and the K approximates the IC in the respective bead refer to Table 2 for guidance EORUDEDNORHDIDISIRE ass 3 The concentration of one labeled protein target In order to derive a K in this type of assay a few criteria should be at least 10X below the concentration of need to be met the other labeled protein tracer If you are using tagged Protein Y untagged Protein Y and tagged Protein X in the competition assay tagged Protein X is the target and tagged Protein Y is the tracer 1 The K needs to be at least 10X higher than the concentration of either tagged protein used in the www perkinelmer com alphatechnology 22 23 Determining K in a Biochemical Assay Competition binding experiment using unlabeled EGF 8 000 E 1 lt t Expected Kd 6 000 5 e 0 1 nM EGFR Fc 1 nM Biotin EGF 4000 2 000 0 L T T T T T 3 11 10 9 8 7 6 5 log unlabeled EGF M Competition binding assay GST HDM2 biotin p53 interaction 400 000 4 Expected Kd GST HDM2 nM ICs uM 300 000 0 61 0 58 z 200 000 0 90 i 1 10 100 000 3 log p53 peptide M Figure 19 Competition binding assay to determine K for a biotinylated EGF EGFR Fc protein protein interaction Streptavidin Donor b
74. used for high throughput screening FUGENE 6 has been observed to work best in the presence of serum and resulting in little or no toxicity Below is an example of a transfection protocol for CHO cells using FUGENE 6 Transfection method for one 100mm Petri dish 0 5 Million CHO cells seeded 16h before 1 Ina 100 mm Petri dish seed 5 x 10 CHO cells 16 hours before transfection Place in a CO incubator 2 Add 576 uL of serum free media to a 2 mL sterile polypropylene tube no antibiotics or fungicide 3 Allow the FUGENE 6 tube to equilibrate to room tem perature before opening Vortex well before opening 4 With a sterile pipet tip add 9 uL of Fugene6 directly to the serum free media Do not touch the sides of the tube Vortex to mix and wait 5 minutes 5 Add 1 6 ug of DNA to the tube Wait 15 min 6 Add the transfection mix to the Petri dish containing the cells and growing media in a drop wise manner over the entire surface Swirl to mix properly 7 24 48h post transfection harvest the cells and prepare the cell lysate We recommend proceeding first with single transfec tions in the cell line of your choice for the two proteins using variable amounts of DNA This will allow confirmation that each plasmid is producing a functional protein in the cells Ideally optimization experiments for DNA amount and post transfection culture time should be conducted prior to performing double transfection experiments
75. was performed in a 384 well OptiPlate for a 25 uL final assay volume according to the following procedure 1 Add 10 uL Anti FLAG Acceptor Beads 20 ug mL final diluted in assay buffer 2 Add 5 uL CHO lysate dilutions in lysis buffer 3 Incubate 15 min at room temperature 4 Add 5 uL of biotin FLAG peptide b nM final in assay buffer Incubate 15 min at room temperature 5 Add 5 uL of streptavidin Donor beads 20 ug mL final diluted in assay buffer 6 Incubate 30 min at room temperature 7 Read on an Alpha capable reader EnVision Multilabel Plate Reader or EnSpire Multimode Plate Reader amp Protein 1 FLAG v FLAG Protein 1 e CHO WT FS PEP SS S Vo a Cells well anes Figure 22 Detection of a FLAG tagged protein in two transfected lysates using an Alpha assay The detection of FLAG tagged Protein 1 with either an N or C terminal tag was done using an AlphaScreen FLAG detection kit Serial dilu tions of the lysates were tested for their ability to compete with the biotin FLAG peptide The amounts of lysate are reported in cells well equivalence 20 000 corresponding to the undiluted lysates Untransfected CHO WT lysates were in cluded as a negative control Here the Protein 1 FLAG transfect C terminal FLAG tag displayed a slightly higher expression level compared to the FLAG Protein 1 transfect N terminal FLAG tag indicating that the FLAG tag in the C terminus may be more optimal
76. xperiment for cell based assays using tagged proteins Titration of cell lysates Pair 4 H Cell lysate Neg Control Figure 29 First experiment for cell based assays using endogenous proteins Antibody selection Negative control single transfected lysate RNAi transfected lysate lysate from uninduced cells or where the interaction has been inhibited www perkinelmer com alphatechnology 36 37 Start up Guide Cell based Assays continued Confirmation that a positive signal is a valid protein protein interaction e For assays studying tagged proteins to confirm that the signal observed is due to the presence of the two specific proteins when possible perform a competition experiment in which increasing concentrations of an untagged recom binant form of one of the two proteins is added to the cell lysates or untagged transfected protein is added by trans fection e For assays studying endogenous proteins to confirm that the signal observed correlates with the amount of double transfected cell lysate perform a titration of the amount of double transfected lysates added to the assay e To further confirm the specificity of the Alpha assay signal reference compounds or peptides known to interfere or modulate the protein protein interaction can be tested for their effect on the assay Microplates for Alpha Assays and Recommended Volumes Scaling the assay volume up or down

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