Technical manual
Contents
1. Baseline 0 000 0 040 i i g a Uy 4 e E 403 Sm 0 085 Wavelength nm 0 03 i j i i i i i i i i 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 S60 Figure 10 NanoDrop absorption spectrum of 4FB modified horseradish peroxidase 220 550 nm 0 66 mg ml sodium phosphate buffer pH 6 0 1 mm path length solulink Catalog A 9302 001 23 g Bovine IgG HRP Conjugate Absorption Spectrum All in One Purified BEU TEENIE IL Mouse HRF gG Allin One nnt Sample 5 Baseline 0 000 ay o a o a lt 1 E E nm eb 0 00 i l l i j i i i i i i i i 220 240 260 260 300 320 340 360 360 400 420 440 460 460 50D Wavelength nm Figure 11 NanoDrop absorption spectrum of All in One IgG HRP conjugate 220 550 nm 0 96 mg ml sodium phosphate buffer pH 6 0 1 mm path length h Concentration of Dilute Antibody Solutions The HRP Antibody All in One Conjugation protocol requires that initial antibody protein concentration be at 4 mg ml in 1 25 ml Many antibody vendors package at significantly more dilute concentrations e g 0 25 to 2 mg ml In these instances IgG samples will need to be concentrated to 4 mg ml and 1 25 ml before proceeding The All in One kit provides a diafiltration filter M W C O 30k for this purpose Figure 12 Carefully follow the instructions below to avoid antibody loss or aggregation when using the filter to concentrate ant2. CHEMICAL HAZARD Some chemicals used can be potentially hazardous and can cause injury or illness e Read and understand the Material Safety Data Sheets MSDS available at Solulink com before you store handle or work with any chemicals or hazardous materials e Minimize contact with and inhalation of chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or clothing For additional safety guidelines consult the MSDS e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s clean up procedures as recommended in the MSDS e Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal F K Catalog A 9302 001 2 sol u 4 Table of Contents Chapter 1 TER OCUICUIO IN i YT E TA 4 a SEF IVa a meaeee A E A Y EFE RR AN EN FFF FFF YN YF FN 4 b PUDO SI il y ET vases snes YU Kr ANTI HNN 4 C oed SC e E RR AFR FR YN CY PA Y FFR NAF Y A FFF YR NE SF NF A 4 d Customer Service and Technical Support uu YY A yd Y GL a FY ND GN ny TG 4 Chapter 2 Overview or Conjugation isie ONC ONN ON NG Geo NWN ON COW CWN Y WON 5 a POU TD CSCI UO aei OD E OD Oa ODNOR Ud FOA DEG DO OO EA IO 5 b HM 9a cei o e1 8 SY Y ER NN FFO FO Y A A RD CH 5 C All in One Conjugation Process SUMMALY ccccssseccccssscceeeeseccccesecceeeeseeceseueceeeeueceessuecetsuneceeseges 8 d Materials P
3. 2 Dirksen A Hackeng T Dawson P 2006 Nucleophilic Catalysis of Oxime Ligations Angew Chem Int Ed 45 7581 7584 3 Dirksen A Dirksen S Hackeng T Dawson P 2006 Nucleophilic Catalysis of Hydrazone Formation and Transimination Implications for Dynamic Covalent Chemistry JIAICIS Communications 4 Lim S Manusu H P Gooley A A Williams K L Rylatt D B 1998 Purification of monoclonal antibodies from ascitic fluid using preparative electrophoresis Journal of Chromatography A Vol 827 Issue 2 11 Pages 329 335 5 Chiodi F Sid n A Osby E 2005 Isoelectric focusing of monoclonal immunoglobulin G A and M followed by detection with the avidin biotin system Electrophoresis Vol 6 Issue 3 124 128 F K Catalog A 9302 001 28 sol u 4
4. column into a 15 ml collection tube not provided 2 Mark the top of the cap using an indelible pen to identify the sample and place a vertical mark on the side of each spin column as shown below HA AA YFFHMW AAAUsA FAYN i me ce a j WN WM MM w Spin column Collection tube 5 ml spin column and 15 ml collection tube 3 Place the assembly into the table top centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor 4 Centrifuge at 1 000 x g for 2 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into the same empty 15 ml collection tube 5 Add 2 5 ml of Buffer B to the top of dry resin bed Centrifuge at 1 000 x g for 2 minute Discard the flow through from the collection tube 6 Repeat step 5 two 2 additional times Place the column back into a new empty 15 ml collection tube not provided N solulink Catalog A 9302 001 7 Remove the cap load the entire antibody HyNic modification reaction 1 25 ml at 4 mg ml to the top of the dry resin bed loosely recap and place the column back into the collection tube 8 Orient the spin column mark outward as before and centrifuge at 1 000 x g for 2 minutes Use an appropriate balance tube opposite the assembly 9 Cap and label the 15 ml collection tube containing the buffer exchanged antibody e Conjugate Formation 1
5. yew Experiment Control Assays Group Window Help GB usc a s Bw v os Bradford Protein Assay o Ca Peten E Setup Eq Template y Reduotion Enoia A Io mw mm 0 o oo Ff Wavelength Combination ILmd Data Mode Absorbance Plate Blank Used Lm 0 356 gt TI standards e Eq se vv iii Unknowns Hat BI of Unknowns CV Dilution Adj Conc gt I Unkno dim Ha 34 afta gt Z Standard Cuvin Ft mE Standard Curve oe PPE EEEE EPEE 0 4 Concentration y A Bor A STDW Standards Concentration vs Mean OD Val 0 011 Figure 7 Print out from a Bradford plate based protein assay F k Catalog A 9302 001 20 sol U n d Using a NanoDrop to Measure Antibody Concentration If an antibody sample is free of protein based carriers e g BSA gelatin or certain interfering preservatives such as thimerosal then a simple non destructive scan of the IgG sample on a NanoDrop spectrophotometer can be used to estimate antibody concentration saving the trouble of conducting a Bradford protein assay to confirm protein concentration To estimate antibody concentration using a NanoDrop M spectrophotometer proceed as follows 1 Turn on the NanoDrop spectrophotometer and click on the NanoDrop icon to launch the software 2 Place a 2 ul drop of molecular grade water on the clean pedestal click OK 3 When the main menu appears select the A280 menu optio
6. 4FB Sulfo N succinimidly 4 formylbenzamide is used at Solulink to form a pre activated high activity form of HRP called 4FB HRP provided in the kit Incubation of HyNic modified antibody with pre activated 4FB HRP in the presence of aniline catalyst F K Catalog A 9302 001 5 sol u 4 leads to rapid and efficient conversion of antibody to conjugate through formation of stable bis arylhydrazone bonds Figure 2 S HyNic Sulfo S 4FB C3H aN4NaO M W 290 2 Cy2HgNO SNa M W 349 25 Figure 1 Structure of S HyNic and Sulfo S 4FB linkers used in conjugating HRP to antibody HyNic 4 FB D N a H a y UN N Y Y aniline catalyst IgG HRP conjugate Uad N SS i uo H H N e 7 H bis arylhydrazone bond Figure 2 Aniline catalyzed formation of IgG HRP conjugate Catalog A 9302 001 6 sol U N 2 Conjugate Purification The efficiency of catalyzed hydrazone bond formation greatly simplifies conjugate purification Because aniline increases both the rate and efficiency of conjugate formation under mild reaction conditions it leads to near quantitative gt 97 conversion of free antibody to conjugate leaving behind only excess 4FB HRP Purification then simply consists of selectively binding the conjugate to a novel filter Q spin filter that exploits biophysical properties of the antibody portion of the conjugate 4 5 while permitting free HRP to flow through unbound In this manner con
7. 50 ml disposable tubes 1 5 microfuge tubes 15 and 50 ml disposable tubes ulink Catalog A 9302 001 9 solu mn Chapter 3 Antibody HRP All in One Conjugation Protocol a IgG Sample Preparation Antibodies come in two physical forms solids or liquids Individual samples can vary significantly in the amount of packaged IgG protein mass and or concentration mg ml We highly recommend that IgG concentrations be confirmed either by a Bradford protein assay or A280 whenever possible The All in One conjugation protocol requires that antibody samples be free of protein carriers such as BSA or gelatin before proceeding A5 milligram quantity of antibody is required to start the procedure Depending on the state of your initial sample solid or liquid proceed as follows Antibody is in Solid Form e g lyophilized powder Resuspend the lyophilized antibody 5 mg free of protein additives gelatin or BSA in 1 25 ml Buffer A to obtain a 4 mg ml solution If the antibody sample contains less than 5 mg per vial e g 1 mg resuspend the requisite number of vials equivalent to 5 mg in 1 25 ml Buffer A to obtain a 4 mg ml solution Proceed to step b Antibody is in Liquid Form e g PBS or TBS Buffer If an antibody sample is in liquid form at 4 mg ml simply transfer 1 25 ml to a labeled microfuge tube 5 mg If the sample is in liquid form at a concentration greater than 4 mg ml transfer a volume equivalent to 5 mg antibody to a labeled mi
8. Spin two dark brown vials containing 4FB modified HRP 5 seconds 1000 x g to collect the contents at the bottom of each tube 2 Transfer the two 1 1 ml 4FB modified HRP aliquots to the tube containing HyNic modified antibody 1 25 ml pipette the mixture 3 5 ml up and down to mix Set the reaction mixture in a dark place or cover with aluminum foil 3 Incubate for 2 h at room temperature f Buffer Exchange Conjugate 1 Ten minutes prior to the end of the conjugation reaction prepare two 5 ml spin columns provided by twisting off the bottom closure and loosening the cap do not remove Place the spin columns into new 15 ml collection tubes not provided 2 Mark the top of the cap using an indelible pen to identify the sample and place a vertical mark on the side of each spin column as shown below see wwe oak a AS Spin column Collection tube 5 ml spin column and 15 ml collection tube 3 Place the two assemblies into a table top centrifuge and orient the vertical mark on the spin columns aiming outward and away from the center of the rotor F K Catalog A 9302 001 13 sol U 4 4 Centrifuge at 1 000 x g for 2 minute Discard the flow through from each collection tube The column matrix will appear white in color Place the columns back into the same empty 15 ml collection tubes 5 Add 2 5 ml of Buffer C to the top of each dry resin bed Centrifuge at 1 000 x g for 2 minutes Discard the flow throug
9. Version 6 01 10 solulink HRP Antibody All In One Large Scale Conjugation Kit Technical Manual Catalog A 9302 001 Note This protocol and any documents linked below can be downloaded from the appropriate category in the Solulink Library at http www solulink com library w solulink Catalog A 9302 001 Disclaimer The products offered here are for research use only Any commercial application will require a license from Solulink The Solulink Conjugation System is patented and has multiple patents pending Please contact Solulink for information regarding licensing information Solulink products and methods may be covered by one or more of the following United States patents Nos 6 686 461 6 800 728 7 102 024 7 173 125 7 462 689 and other pending patent applications Information in this manual is subject to change without notice and does not constitute a commitment on the part of Solulink Inc It is supplied on an as is basis without any warranty of any kind either explicit or implied Information may be changed or updated in this manual at any time This document may not be copied transferred reproduced disclosed or duplicated in whole or in part without the prior written consent of Solulink Inc This documentation is proprietary information and protected by the copyright laws of the United States and international treaties The manufacturer of this documentation is Solulink Inc Safety Information WARNING
10. crofuge tube and add Buffer A to obtain a 4 mg ml solution If a sample is at a concentration less than 4 mg ml concentrate the sample to 1 25 ml and 4 mg ml using any suitable ultra filtration spin filter e g Amicon or VivaSpin as described in the Appendix A concentration filter is provided with this kit Proceed to step b b Buffer Exchange IgG 1 Prepare a 5 ml spin column provided by twisting off the bottom closure and loosening the cap do not remove Place the spin column into a 15 ml collection tube not provided 2 Mark the top of the cap using an indelible pen to identify the sample and place a vertical mark on the side of each spin column as illustrated on the next page F K Catalog A 9302 001 10 sol u 4 TEETE VALVE ety i m amp 5 WWW Spin column Collection tube 5 ml spin column and 15 ml collection tube 3 Place the assembly into the table top centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor 4 Centrifuge at 1 000 x g for 2 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into the empty 15 ml collection tube 5 Add 2 5 ml of Buffer A to the top of dry resin bed Centrifuge at 1 000 x g for 2 minutes Discard the flow through from the collection tube 6 Repeat step 5 two 2 additional times Place the column back into a new empt
11. endix F K Catalog A 9302 001 16 sol u 4 Chapter 4 Appendix a Monoclonal lIgG HRP Conjugate An Example Greater than 97 5 pure HRP GK1 5 mAb conjugate Less than 2 5 by integrated area is free antibody ko FF ie u s Y we W W H w a Ud a 43 bere ae b i 9 J 7 r Iy 4 a Y a P a F a 4 as lt Free HRP en a Pi mh _ La b Figure 5 Coomassie stained 4 12 SDS PAGE gels illustrating typical conjugation results Horseradish peroxidase is a 44 kD highly glycosylated protein that migrates as a broad high M W band when the protein sample is not heated 70 C before loading on the SDS PAGE gel Lane 1 Protein M W Marker Lane 2 4FB HRP 1x LDS sample buffer unheated sample 7 5 ug Lane 3 4FB HRP 1x LDS sample buffer heat treated 70 C 4 min 7 5 ug Lane 4 GK1 5 mAb 1x LDS sample buffer unheated sample 1 5 ug Lane 5 GK1 5 mAb 1x LDS sample buffer heat treated 70 C 4 min 1 5 ug Lane 6 GK1 5 HRP crude conjugation rxn 1x LDS sample buffer unheated sample 7 5 ug Lane 7 GK1 5 HRP purified conjugate 1x LDS sample buffer unheated sample Lane 8 GK1 5 HRP purified conjugate 1x LDS sample buffer heat treated 70 C 4 min Lane 9 GK1 5 HRP Q filter 1 flow through 20 uL 1x LDS sample buffer unheated sample Lane 10 GK1 5 HRP Q filter 1 flow through 20 uL 1x LDS
12. h from each the collection tube 6 Repeat step 5 two 2 additional times Place the column back into a new empty 15 ml collection tube not provided 7 Remove the caps load 1 75 ml conjugate onto each spin column Section e step 3 to the top of the dry resin bed loosely recap and place the columns back into their collection tubes 8 Orient the spin column mark outward as before and centrifuge at 1 000 x g for 2 minutes 9 After centrifugation add 3 5 ml Buffer C to the bottom of each collection tube containing the conjugate 5 25 ml volume in each collection tube and pipette up and down to mix Set both collection tubes aside containing the conjugate in a dark place or cover with aluminum foil g Q Spin Filter Purification 1 Pre wet a Q spin filter by adding 1 5 ml Buffer C to the top of the unit see filter below and incubate for 2 minutes ee Q O Spin Filter Q Collection Tube 2 Place the spin filter assembly into a table top centrifuge and orient the letter Q towards the center of the rotor spin at 500 x g for 4 minutes discard the flow through and place the filter back into the empty collection tube 3 Load one half of the antibody HRP conjugate volume 5 25 ml section f step 9 to the top of the filter unit and incubate for 2 minutes 4 Place the oriented and balanced assembly in the table top centrifuge and spin at 500 x g for 4 minutes F K Catalog A 9302 001 14 sol
13. he top of the cap using an indelible pen to identify the sample and place a vertical mark on the side of the spin column as shown below Spin column Collection tube 5 ml spin column and 15 ml collection tube F K Catalog A 9302 001 15 sol U 4 3 Place the balanced assembly into a table top centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor 4 Centrifuge at 1 000 x g for 2 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into the same empty 15 ml collection tube 5 Add 2 5 ml of PBS buffer to the top of dry resin bed Centrifuge at 1 000 x g for 2 minute Discard the flow through from the collection tube 6 Repeat step 5 two 2 additional times 7 Place the spin column into a new 15 ml collection tube not provided 8 Remove the cap load 1 5 ml eluted conjugate Section g step 12 to the top of the dry resin bed loosely recap and place the column back into the collection tube 9 Orient the spin column mark outward as before and centrifuge at 1 000 x g for 2 minutes Use an appropriate balance tube opposite the assembly 10 After centrifugation transfer the purified conjugate solution 1 5 ml from the bottom of the collection tube to a new 1 5 ml tube and label appropriately 11 Measure the final conjugate protein concentration using a Bradford or BCA protein assay see App
14. hromatography or other methods make sure to properly mix the antibody HyNic reaction mixture use a Calibrated P 10 pipette to insure accuracy of small volumes remove all non protein amine contaminants such as glycine or Tris before modification keep and store S HyNic sealed in the aluminum solulink initial antibody concentration was too low or too high Low conjugate and or low spin column recovery volume antibody recovery j Component Stability on Storage Component Unopened Kit S HyNic HRP Antibody Conjugate All other kit components Catalog A 9302 001 24 h after re suspending S HyNic in DMF pouch provided that contains dessicant measure the initial antibody concentration before proceeding Bradford or NanoDrop concentrate or dilute the antibody sample into the recommend range 4 5 mg ml and 25 ul before proceeding use a properly calibrated variable speed centrifuge Follow recommended spin speed time Altered spin speeds can adversely compromise protein and or volume recovery Storage Condition Refrigerated 2 8 C Keep in sealed aluminum pouch provided 2 8 C Room temperature Refrigerated 2 8 C in final conjugate solution Refrigerated 2 8 C 50 glycerol Refrigerated 2 8 C solulink k References 1 Dirksen A Hackeng T Dawson P 2007 Nucleophilic Catalysis of Oxime and Hydrazone Reactions by Aniline ACS Poster
15. ibody Note dilute antibody solutions require 5 milligrams of starting antibody e g 2 ml 2 5 mg ml Concentrator body hea f Wr lt Filtrate tube Figure 12 Diafiltration spin filter used for concentrating dilute antibody samples prior to the start of All in One conjugation protocol F K Catalog A 9302 001 24 sol u n Antibody Concentration Protocol Note the diafiltration spin filter provided is made to contain and process a maximum volume of 500 ul To process sufficient volume of dilute antibody solution equivalent to 5 milligrams multiple loadings and spins will be required 1 2 3 4 5 6 7 Open the lid of the diafiltration spin filter device provided Transfer 500 ul of dilute protein solution to the center of the filter cup Close the lid and orient the spin filter in the centrifuge so that the volume marker faces toward the center of the centrifuge rotor Use an appropriate balance tube opposite the spin filter Centrifuge for 2 minutes 5 000 x g Do not centrifuge for a longer periods of time to avoid antibody aggregation Open the filter unit and visually note the volume remaining Bring the volume back in the spin filter concentrator body to 500 ul by adding additional dilute antibody solution Pipette the solution 500 ul up and down at least 20 times to fully resuspend the concentrated antibody away from the filter s surface Repeat steps 4 and 5 until a volume eq
16. jugate is eluted in highly purified form with high yield 4 to 5 milligrams Y o lgG HRP conjugate free excess un conjugated 4FB modified HRP a d Bind conjugate to O 5pin Filter s sy _ Bind conjugate to O Spin Filter n lt _ free HRP passes through O Spin Filter Wash filter and discard wash spin cn Elute conjugate from filter spin L a Pure IgG HRP conjugate Figure 3 Q spin filter purification of HRP lgG conjugates Ga K Catalog A 9302 001 7 sol u 4 c All in One Conjugation Process Summary Y Buffer Exchange IgG lt v Spin column 3 min S HyNic linker HyNic modify IgG Tg 2h HyNic gt Excess S HyNic Buffer Exchange IgG _ g Spin column 3 min fwr 4FB 4FB HRP Conjugate Formation Fm Excess 4FB HRP Lb Spin column Conjugate Purification cy O Spin Column 45 min CS T JL i Spin column LA Purified IgG HRP conjugate Figure 4 Summary of conjugation process e kK Catalog A 9302 001 8 sol U nN d Materials Provided and Storage Conditions S HyNic 1 x 500 ug Keep refrigerated within desiccated pee ia e e Additional Materials Required But Not Provided Bradford Protein Assay Reagents verification of final conjugate concentration Standard UV VIS or NanoDrop Spectrophotometer Pipettes P 10 P 100 P 1000 and tips Table Top Centrifuge e g variable soeed capable of handling 15 and
17. late Procedure Required Materials Bradford Reagent Bio Rad Hercules CA Cat 500 0006 96 well microtiter plate standard flat bottom PBS phosphate buffered saline P 200 and P 1000 pipettes Bovine IgG Antibody Standard 2 mg ml Pierce ThermoFisher Cat 23212 Molecular grade water Assay Protocol 1 Prepare 2 ml of a Bradford working solution by adding 400 ul dye reagent to 1600 ul molecular grade water 1 4 ratio 2 Prepare the following protein dilution standards and blank as follows Add 160 ul 2 mg ml bovine IgG standard to 240 ul PBS 0 8 mg ml standard Add 150 ul 0 8 mg ml standard to 50 ul PBS 0 6 mg ml standard Add 75 ul 0 6 mg ml standard to 25 ul PBS 0 4 mg ml standard Add 50 ul 0 4 mg ml standard to 50 ul PBS 0 2 mg ml standard Add 50 ul 0 2 mg ml standard to 50 ul PBS 0 1 mg ml standard Add 50 ul PBS buffer blank 3 Pipette 5 ul of each standard and blank along with duplicates of appropriately diluted antibody sample into separate microtiter wells 4 Add 100 ul of previously diluted dye reagent 1 4 to each well and mix thoroughly Always replace pipette tips between additions 5 Incubate at room temperature for 5 10 minutes but no more than 60 minutes 6 Measure absorbance at 595 nm on a suitable microtiter plate reader F K Catalog A 9302 001 19 sol u 4 7 A typical Bradford plate assay result from a commercial plate reader is illustrated in Figure 7 ii Ele Edit
18. llustrated on the following page F K Catalog A 9302 001 21 sol U 4 Example A mouse IgG sample at 1 mg ml in PBS 100 ul was scanned as described and its concentration confirmed using equation 1 below i i F i i 1 ieee eM 400 311 don 330 340 S50 elength rni Figure 8 A mouse IgG sample 100 ul 1 mg ml in PBS pH 7 2 scanned on the NanoDrop as described in the text Sample Calculation Equation 1 A280 E196 value x 10 mg ml protein concentration mg ml Example Mouse IgG 1 mg ml Fig 8 A280 reading from scan in Figure 8 1 34 Antibody E1 value Table 1 14 00 A280 E1 bovine IgG x 10 mg ml protein concentration mg ml 1 34 14 00 x 10 mg ml 0 96 mg ml Table 1 Mass extinction coefficients E1 used for calculating antibody concentration The E1 is the A280 of a 10 mg ml solution 1 cm path length solulink Catalog A 9302 001 22 e HRP Absorption Spectrum Unmodified Horseradish peroxidase HRF Unmoditied 0 66 mg ml Sampla 11 Baseline 0 000 1 mrm Absorbance wt ra i 0015 i i i i i I i 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 550 HT Wavelength nm uJ Eu EU Figure 9 NanoDrop absorption spectrum of unmodified horseradish peroxidase 220 550 nm 0 66 mg ml sodium phosphate buffer pH 6 0 1 mm path length f 4FB modified HRP Absorption Spectrum 4FB HRP 0 66 mg ml Sample 15
19. n Note do not use the UV VIS menu option on the NanoDrop to read an antibody sample 4 After the A280 menu appears click off the 340 nm normalization option using the mouse Note some instruments do not use this normalization feature in which case this step can be ignored 5 In the window labeled Sample Type select Other protein E1 option from the pull down menu Enter the appropriate E196 value Table 1 on the next page corresponding to your particular antibody sample type For example 14 00 for mouse IgG 6 Blank the NanoDrop spectrophotometer by placing a 2 ul drop of the appropriate sample buffer e g PBS and click on the Blank icon 7 Immediately re click the Measure icon to validate the baseline i e flat across the bandwidth Clean the pedestal and repeat if necessary until a flat baseline is obtained Note sometimes air bubbles can become trapped on the pedestal during sample loading and cause baseline offsets If necessary remove air bubbles and rescan to insure a proper baseline 8 Place a 2 Mul volume of antibody solution on the clean pedestal and click the Measure icon Wait until the spectrum 220 350 nm appears in the window Note for precious or limited samples the majority of the 2 ul aliquot can be recovered from the pedestal 9 Record antibody concentration directly from the NanoDrop display window mg ml Alternately calculate the antibody concentration manually as i
20. n experience allowing them to prepare customized high purity ready to use HRP antibody conjugates within a single day d Customer Service and Technical Support Additional technical information can be found at Telephone Email 1 888 625 0670 Toll Free Solulink Solulink com Fax Address 1 858 625 0770 Solulink The Conjugation Company 9853 Pacific Heights Blvd Ste H San Diego CA 92121 K Catalog A 9302 001 4 sol U 4 Chapter 2 Overview of Conjugation a Product Description Each HRP Antibody All in One Conjugation Kit provides all the necessary components to produce one 1 highly purified HRP lgG conjugate The kit requires a user to provide 5 milligrams of starting IgG antibody Any suitably purified monoclonal or polyclonal antibody regardless of species origin or IgG subclass can be conjugated and purified within 5 hours 60 minutes hands on The components of this kit feature a stable high activity pre activated horseradish peroxidase gt 250U mg and a novel Q spin filter that delivers purified conjugate in high yield approx 4 5 mg Conjugates are gt 95 pure free of both residual antibody and un conjugated HRP Conjugates produced are guaranteed to provide optimum signal to noise in sensitive downstream applications All in One conjugation kits are based on Solulink s patented HydraLink chemistry This chemistry relies on a specific reaction between an aromatic hydrazine HyNic and an aromatic aldeh
21. rovided and Storage Conditions eesesssssessssrereserrssrreressrrrssereresrrresrreresrrreserereserresene 9 e Additional Materials Required But Not Provided sssssssusessresesrersssrereseresesrrresrrrrssrrresereresreresene 9 Chapter 3 Antibody HRP All in One Conjugation Protocol sessesessssessssessssessssesesseosssssesesssoseseoseee 10 a EG ample Prepar atl OW assistance a E 10 b Bu er EX N nE 1 a E E DEON ORO 10 C PONEC MOOI IE e E RNA EW ET HNNAN FYNN Y nae 11 d Burer ECHO Ol Gates i DA Cd S 12 e i a 8 42 eU 9 d gt uc 1 ET nian noeteneieotienaboadesastaa sitar hbotaderiaambiiadasdunatteadiaaa bes mehiaceineratinnnns 13 f BUI eh Exchange Conjug ale SEF Y GE E OH FE 13 g PA FU Gi PILI OU OT ET E A S 14 h Buffer Exchange Purified ConJugdL amp Ge GU GN Y GEI dd GO GWA Yn yd 15 Chapter 4 Appendik HF Y RH CH HR REFER EL NI FFF ELF saoaees 17 a Monoclonal IgG HRP Conjugate An Exam ple cccccccssssccccessececeeesececseeceeseeseeesausecesseneceesenes 17 b Direct ELISA Assay Using IgG HRP All in One Conjugate ccccccessecceceseceeeeseceeaeecesseneceesenes 18 C Bradiord Protein AS SAV RR FERRY A E S 19 d Using a NanoDrop to Measure Antibody Concentration ccccccecsesseccecceccesseeseeseesseseesceseesees 21 e HRP Absorption Spectrum Unmodified Horseradish peroxidase ccccccccssessseeeseseseeneeeseenes 23 f 4FB modified HRP Absorption SpectrUmM ssssesse
22. sample buffer heat treated 70 C 4 min if k Catalog A 9302 001 17 sol u n b Direct ELISA Assay Using IgG HRP All in One Conjugate Direct ELISA Standard Curves Lyg ml 0 5 ug ml 0 25 pg ml E c o in o 2 gt gt un c o o T a db a o c a Antigen Concentration ng ml Figure 6 Direct ELISA curves generated using an HRP conjugate made with the All in One kit A mouse anti FITC monoclonal antibody was conjugated to HRP as described in the manual Antigen consisting of FITC labeled BSA FITC MSR 2 was coated on plates in a 2 fold dilution series 100 ul 500 250 125 62 5 31 25 15 625 7 8 3 90 and 1 95 ng ml using standard methods Immobilized antigen was then detected at 3 different conjugate concentrations 1 ug ml 0 5 ug ml 0 25 ug ml using TMB substrate 20 minutes 450 nm on a Molecular Devices plate reader Catalog A 9302 001 18 sol U N c Bradford Protein Assay Solulink highly recommends that whenever IgG is not limiting or its concentration source or quality are unknown that the sample be assayed for initial protein concentration using a Bradford protein assay prior to conjugation The starting quality and quantity of antibody is critical to the success of the procedure A reference assay protocol is provided for measuring antibody or conjugate protein concentrations using Bradford protein reagents not provided in this kit Bradford Microtiter P
23. sessenserreresrrrssrreresrresssrrereserersseeresererssrereserereseeee 23 g Bovine IgG HRP Conjugate Absorption Spectrum All in One Purified neeese 24 h Concentration of Dilute Antibody SOLUTIONS ssosssseseesesreeserreresrerrssrreresrersssreresreressrereserersseeee 24 i i 910 8 Lc a 6 9 11 UO e E E TA 26 j Component Stability on Storage dieu iu ddaa due GOD a Ond nd UD OU UAN iddn ANY L nwn Gwnai 27 k RETCVCING EF REF NEI FR RIFAU FEARN FI FFF FA FFA RR EFA RAF FFF 28 ev K Catalog A 9302 001 3 sol U 4 Chapter 1 Introduction a User Manual This manual provides instructions for using the HRP Antibody All in One Large Scale Conjugation Kit This chapter contains the following sections Purpose of Manual Intended Users Customer Service and Technical Support b Purpose of Manual The purpose of this manual is to provide the user with the necessary instructions and reagents to produce one 1 HRP antibody conjugate Use of the kit results in e The modification of a user supplied IgG antibody 5 milligrams using S HyNic linker e The conjugation of HyNic modified IgG with 4FB HRP resulting in formation of an HRP antibody conjugate e Spin filter isolation of highly purified IgG HRP conjugate yielding approximately 4 5 milligrams of material free of residual enzyme and antibody c Intended Users The HRP Antibody All in One Kit is designed for users with minimal or no conjugatio
24. u n 5 Load the second half of the antibody HRP conjugate volume 5 25 ml section f step 9 to the top of the same filter unit and incubate for 2 minutes 6 Place the oriented assembly in the centrifuge and spin at 500 x g for 4 minutes discard the flow through from the collection tube and place the Q spin filter back into the same empty collection tube Note a brown colored conjugate will be visible on the top of the Q membrane 7 Add 5 ml Buffer C to the filter unit orient in the table top centrifuge and spin at 500 x g for 4 minutes discard the flow through from the bottom collection tube and place the filter back into the same empty collection tube 8 Repeat step 7 three 3 additional times 9 Remove the Q spin filter unit from its collection tube and place it into a new collection tube provided 10 Add 0 5 ml Buffer D to the top of the O spin filter containing the tightly bound brown conjugate and incubate for 5 minutes on the bench top 11 Place the oriented and balanced assembly in a table top centrifuge and spin at 500 x g for 4 minutes to elute 12 Repeat step 10 and 11 two 2 additional times Total elution volume will be 1 5 ml Set the collection tube aside on the bench h Buffer Exchange Purified Conjugate 1 Prepare a 5 ml spin column provided by twisting off the bottom closure and loosening the cap do not remove Place the spin column into a 15 ml collection tube not provided 2 Mark t
25. uivalent to 5 milligrams is processed Transfer the concentrated IgG solution equivalent to 5 milligrams to a new 1 5 ml microfuge tube and bring the volume to 1 25 ml using Buffer A to achieve a 4 mg ml IgG solution You may now proceed with the conjugation protocol F K Catalog A 9302 001 25 sol u 4 i Troubleshooting Guide Problem Poor conjugate yield Poor conjugate yield Poor HyNic modification Poor HyNic modification Catalog A 9302 001 Possible Cause initial antibody concentration and volume were incorrect or unknown Starting antibody concentration and volume are incorrect or unknown presence of protein carrier e g BSA or gelatin is contaminating the antibody sample improper mixing of HyNic reaction components presences of amine contaminants improper storage of S HyNic reagent can lead to hydrolysis of this NHS 26 Recommended Action whenever possible verify the original starting antibody concentration using a Bradford protein TM assay or NanoDrop to assure efficient conjugation concentrate or dilute the antibody sample to be conjugated into the required range 4 5 mg ml and 25 ul preservatives can interfere with the accuracy of a Bradford protein assay Remove all interfering preservatives such as thimerosal or proclin before performing a Bradford protein assay remove and purify away all protein carriers such as BSA or gelatin using affinity c
26. y 15 ml collection tube not provided 7 Remove the cap load the antibody sample 1 25 ml at 4 mg ml to the top of the dry resin bed loosely recap and place the column back into the collection tube 8 Orient the spin column mark outward as before and centrifuge at 1 000 x g for 2 minutes Use an appropriate balance tube opposite the assembly IMPORTANT rotor speed should be set to 1000 x g RCF and not 1000 x rpm RPM The volume recovered should always be approximately the same volume that was loaded on the spin column e g 1 25 0 2 ml If the recovered volume is low the centrifuge may require recalibration If volume is low re centrifuge at the appropriate speed in an attempt to recover the full volume i e 1 25 ml 9 Cap and label the 15 ml collection tube containing buffer exchanged antibody c HyNic Modify IgG 1 Add 100 ul DMF to S HyNic reagent vial Pipette the solution up and down to resuspend the reagent pellet F K Catalog A 9302 001 11 sol U 4 2 Add 58 ul dissolved S HyNic reagent to the antibody solution 1 25 ml 4mg ml Pipette the solution up and down to mix 3 Incubate the reaction for 2 h at room temperature Set the mixture in a dark place or cover with aluminum foil d Buffer Exchange IgG 1 Ten minutes before the end of the IgG modification reaction prepare a 5 ml spin column provided by twisting off the bottom closure and loosening the cap do not remove Place the spin
27. yde 4FB leading to formation of a stable bis arylhydrazone bond HydraLinK conjugation chemistry is capable of efficiently converting nearly 100 of an antibody to its conjugate form This efficiency is made possible through the recent discovery that aniline rapidly catalyzes hydrazone bond formation 1 2 3 Aniline s ability to increase both the rate and efficiency of conjugate formation under mild reaction conditions yields reproducible and quantitative conversion of free antibody to conjugate Complete conversion of conjugate greatly simplifies downstream purification Conjugate purification consists of selectively binding conjugate to a novel Q spin filter membrane that allows excess HRP to flow through unbound This spin filter provides high purity without sacrificing yield Conjugates made with these kits are compatible with all downstream applications requiring high immunological specificity and sensitivity such as Westerns ELISAs or IHC Finally each kit yields between 4 and 5 mg of highly purified HRP antibody conjugate b All in One Technology 1 Conjugation Chemistry HydraLink chemistry is based on the use of two complementary heterobifunctional linkers S HyNic and Sulfo S 4FB Figure 1 S HyNic Succinimidly 6 hydrazino nicotinamide is used to modify and incorporate protected aromatic hydrazines HyNic groups into the antibody via acylation of lysine residues In a similar fashion a second linker known as Sulfo S
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