Technical manual
Contents
1. collection tube 2 Place the assembly in the centrifuge and orient the letter Q towards the center of the rotor spin at 2 000 x g for 4 minutes discard the flow through from collection tube and place the filter back into the empty collection tube Load the R PE Antibody conjugate 450 from the previous section to the top of the filter unit and allow to incubate for 2 minutes with the filter F Catalog A 9001 006 16 sol U 4 Place the oriented assembly in the centrifuge and spin at 2 000 x g for 4 minutes discard the flow through from the collection tube and place the filter back into the bottom empty collection tube Note a highly pink color will appear bound to the top of the Q filter membrane 5 Add 400 uL Buffer E to the filter unit orient in the centrifuge and spin at 2 000 x g for 4 minutes discard the flow through from the bottom collection tube and place the filter back into the empty collection tube 6 Repeat step 5 two 2 additional times 7 Remove the top filter unit from the collection tube and place it into a new collection tube provided 8 Add 100 uL Buffer F to the top of the pink colored filter and incubate for 5 minutes on the bench top 9 Place the oriented assembly in the centrifuge and spin at 2 000 x g for 4 minutes 10 Open the lid of the spin filter and add an additional 50 uL Buffer F to the top of the filter and spin the oriented assembly for a2. In a second purification stage a novel Q spin filter selectively binds conjugate but excludes free IgG from binding based on well known biophysical properties of nearly all antibodies This novel dual purification process completely avoids costly and time consuming column chromatography procedures while removing all traces of free IgG and virtually all free R PE The result is a high activity conjugate consisting primarily of 1 1 heterodimer 28096 with minor residual quantities of 1 2 heterodimer 20 in Catalog A 9001 006 6 sol u high yield see Appendix All purified conjugates are ready for the most demanding downstream applications IgG R PE residualfree R PE residual free IgG Co Bind crude conjugation ee mixture to magnetic beads Wash away residual R PE i Elute from beads residual R PE 214 r X v E Bind conjugate to Q spin filter C spin gt Wash away residual IgG Elute conjugate IgG R PE conjugate Figure 3 Two stage purification of IgG R PE conjugate Catalog A 9001 006 7 sol u Ini C All in One Conjugation Process Summary pin Buffer exchange Prepare IgG Tor Modification il E T wired cap spin column 5 7 min HyNic linker Vo Modify IgG w HyNic 120 min Y En Buffer exchange Buffer Exchange HyNic igG 4 14 w yellow cap spin column 3 min Y HyNic IgG HyNic IgG Form conjugate 120 min Y conj
3. 600 625 650 Wavelength nm Sample Baseline Ls 2 4 35 2 191 0 67 80 24 Figure 8 Absorption spectrum of H phycoerythrin 250 650 1 38 mg ml sodium phosphate buffer pH 8 0 solulink Catalog A 9001 006 24 E R PE Absorption Spectrum 4FB modified sample ID 4FB PE 00 Absorbance Normalize Off HiAbs Dm Sample b Baseline 0 000 Al 230 1 200 8 6 E 4 566 nm 1 145 0 00 0 10 A i i i i i i t i i i i i 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 35 2 191 0 67 80 24 Wavelength nm Figure 9 Absorption spectrum of 4FB modified R phycoerythrin 250 650 nm 1 38 mg ml sodium phosphate buffer pH 8 0 F Concentration of Dilute Antibody Solutions The R PE Antibody All in One Conjugation protocol requires that initial IgG sample be at a protein concentration of 1 mg mL and 100 ul For IgG samples at concentrations significantly less than 1 mg mL e g 0 1 to 0 5 mg mL the IgG must first be concentrated to 1 mg mL and 100 uL using a suitable ultra filtration spin filter before proceeding Although the All in One kit does not provide an ultra filtration spin filter it does provide a recommended reference protocol for concentrating dilute antibody solutions Figure 10 We routinely employ a VivaSpin 500 M W C O 30 kD from Sartorius Stedim U
4. mg mL using any suitable ultra filtration spin filter e g Amicon or VivaSpin 500 as described in the Appendix A concentration filter is not provided with this kit Proceed to step b B Buffer Exchange IgG 3 minutes 1 Prepare a spin column red cap by twisting off the bottom closure and loosening the red cap do not remove Place the spin column into a collection tube provided Ifi kc Catalog A 9001 006 10 solu Ir 2 Mark the top of the red cap using an indelible pen to identify the sample and place a vertical mark on the side of each spin column as shown next Label lid w antibody ID i gt Place pen mark on Side of spin column Collection tube 3 Place the assembly into the centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor 4 Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided 5 Remove the red cap load the antibody sample 100 uL at 1 mg mL to the top of the dry resin bed loosely recap and place the column back into the collection tube 6 Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes Use a balance tube opposite the assembly Note Rotor speed should be set to 1500 x g RCF and not 1500 x rpm RPM The volume recover
5. pre activated 4FB PE in the presence of aniline as catalyst leads to rapid and efficient conversion of the antibody to conjugate through formation of stable bis arylhydrazone bonds Figure 2 db dh d S HyNic Sulfo S 4FB B Ci 3 H44N4NaO M W 290 2 C 2 Hag NOgSNa M W 349 25 Figure 1 Structure of S HyNic and Sulfo S 4FB linkers used to conjugate R PE to antibody e Catalog A 9001 006 5 sol u 4 B ee d On 80 y C N T Ee N ii H aniline catalyst bis aryl hydrazone bond Figure 2 Conjugation chemistry used in linking IgG to R PE Purification Technology This All in One kit uses a two stage purification process to generate highly purified R PE antibody conjugates as illustrated in Figure 3 Kits have been engineered to consistently produce a 1 1 heterodimer product starting with any mammalian antibody The perfect heterodimer ratio 1 1 is made possible by the recent discovery that hydrazone bond formation is rapidly and efficiently catalyzed in the presence of aniline Aniline facilitates hydrazone bond formation between biomolecules at low protein concentrations and equivalent mole ratios both of which are necessary for the consistent formation of a heterodimer In the first stage of the purification process the 1 1 heterodimer and any excess un conjugated IgG are purified away from excess 4FB PE using a magnetic affinity matrix or bead
6. 9 Chapter 3 All in One Conjugation Protocol eene 9 A IgG Sample Preparation 5 10 D B ffer Exchange lgG 10 SMEs rlehueerd chr 12 D BufferExchange MINUTES ate enn re eer S 12 L Conjugate Formation 2 13 F Conjugate Purification Stage 1 Affinity Isolation 1 34 13 G Buffer Exchange Conjugate 3 minutes 15 Conjugate Purification Stage 2 Spin Filter 35 min eese 16 L Buffer Exchange Conjugate 9 minutes iub a GE UEM REN EE 17 Chapter 4 ADDODUDG o earn 18 A Human IgG R PE Conjugate Monoclonal An 18 D Broad PrO CII ASSQU 19 C Using a NanoDrop to Measure Antibody Concentration seen 21 D R PE Absorption Spectrum Unmodified R PE cccccccccsssseccceeeesesecceeeeeesecceseeeeeeeeeeeueneeeeseeeeees 24 E R PE Absorption Spectrum 4FB Modified ccccccccessccccccessseccecceeeececesseeeseeeeeeueneeeeseueneceeeeeaenes 25 Concentration of Dilute Antibody 25 G Troubleshooting Guide NER 27 RETON O e 28 Tab
7. A Product Description Each R PE Antibody All in One Conjugation kit is designed to produce two high quality ready to use R PE antibody conjugates Each conjugation requires 100 ug of user supplied mammalian IgG from any species The kit is designed to produce heterodimer conjugates 1 1 R PE to IgG ratio devoid of virtually all free R PE or residual IgG Any suitably purified monoclonal or polyclonal antibody free of protein carriers or stabilizers e g BSA or gelatin can be used to form the conjugate The kit employs a dual purification strategy using magnetic affinity beads and a Q spin filter Conjugates produced are fully compatible with all demanding downstream applications including flow cytometry and or sensitive microarray detection Each kit contains sufficient reagents to produce two ready to use R PE antibody conjugates 7125 ug each B All in One Conjugation Technology Conjugation Chemistry HydraLinK chemistry is based on the use of two complementary heterobifunctional linkers S HyNic and Sulfo S 4FB Figure 1 S HyNic Succinimidyl 6 hydrazino nicotinamide is first used to modify and incorporate protected aromatic hydrazines HyNic groups into the antibody via acylation of lysine residues In a similar fashion the second linker Sulfo S 4FB Sulfo N succinimidyl 4 formylbenzamide is used by oolulink to form a pre activated form of R PE called 4FB PE provided in the kit Incubation of the HyNic modified antibody with
8. G Sample Preparation 5 minutes Antibodies come in two physical forms solids or liquids Individual samples can vary significantly in the amount of packaged lgG protein mass and or concentration mg ml We highly recommend that IgG concentrations be confirmed either by Bradford protein assay or A280 whenever possible The All in One conjugation protocol requires antibody samples to be free of protein carriers such as BSA or gelatin before proceeding A 100 ug mass 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 100 ug free of protein additives gelatin or BSA in 100 ul Buffer A to obtain a 1 mg mL solution If the antibody sample contains less than 100 ug per vial e g 50 ug resuspend the antibody in the requisite number of vials equivalent to a 100 ug in 100 ul Buffer A to obtain a 1 mg mL solution Proceed to step b Antibody is in Liquid Form e g PBS or TBS Buffer lf the antibody sample is in liquid form at 1 mg ml simply transfer 100 ul to a labeled microfuge tube 100 ug If the sample is in liquid form at a concentration greater than 1 mg ml transfer a volume equivalent to 100 ug antibody to a labeled microfuge tube and add Buffer A to obtain a 1 mg ml solution If a sample is at a concentration less than 1 mg ml concentrate the sample to 100 uL at 1
9. SA for this purpose Carefully follow the instructions provided below to avoid irreversible aggregation and loss of IgG on the filter surface Note dilute antibody solutions may require 125 ug mass of antibody e g 500 ul 0 25 mg ml since ultra filtration filters typically recover about 80 of input antibody Concentrator body Figure 10 Ultra filtration spin filter used for concentrating dilute antibody samples prior to the start of All in One conjugation protocol Catalog A 9001 006 25 sol u Filtrate tube IgG Concentration Protocol Note these ultra filtration spin filters are made to contain and process a maximum volume of 500 ul or less If a volume greater 0 5 ml is to be concentrated multiple loadings will be required 1 Open the lid of a filtration spin filter device 2 Transfer 500 ul or less of dilute protein solution equivalent to 125 ug antibody to the center of the filter cup 3 Close the lid and orient the spin filter in the centrifuge so that the volume markers face toward the center of the centrifuge rotor Use an appropriate balance tube opposite the spin filter 4 Centrifuge for 2 minutes 5 000 x Note never increase the centrifugation time 5 Open the filter unit and pipette the remaining volume up and down gently 20 times to resuspend the antibody back into solution and away from the filter surface Note take care not to touch
10. Version 08 31 2012 solulink R PE Antibody All In One Conjugation Kit Technical Manual Catalog A 9001 006 Note This protocol and any documents linked below can be downloaded from the appropriate category in the Solulink Library at Catalog A 9001 006 1 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 CHEMICAL HAZARD Some chemicals used can be potentially haza
11. cal Bradford assay result from a commercial plate reader is illustrated in Figure 6 on the next page Elle Edit View Experiment Contro assays Group Window GB 255 c rea es e gt ce Br adiford Protein Assay v Paten Setup ES Template E Reduction itia L a PIates 1 1 2 3 4 5 amp 7 g 10 11 Ta Plate Last Read 359 12 15 2008 I m oc P Wievelength Comberation Data Mode Absorbance Plate Blank Used Lm 0 356 b FTE standarcs 2 5 8l EFT Unknowns He 24 afta Unknowns Sample Wells OD Values Concentration Mean Conc Std Dew CV Dilution Adj Conc i 1 1299 250 8 oo 1 208 2 Bio 020 290 000 10 7 Cw Dam 0 000 00 1 0 0 45 b 10 24 Zbsmndwdcuveit Ft iE al Standard Curve Mean OD value 01 02 03 0 4 0 5 06 0 7 08 Concentrabon amp B E o STA Standards Concentration Mean OD Val 0 011 1 Figure 6 Print out from a Bradford plate based protein assay C Using a NanoDrop to Measure Antibody Concentration If the 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 accurately determine its concentration To estimate antibody concentra
12. ed should always be approximately the same volume loaded on the spin column e g 100 10 uL If the recovered volume is low the centrifuge may require recalibration It is recommended to re centrifuge at the appropriate speed in an attempt to recover the full volume i e 100 LL 7 Transfer the solution from the bottom of the collection tube to a 1 5 mL tube and label appropriately solulink Catalog A 9001 006 C HyNic Modification of IgG 21 1 Add 20 ul DMF to S HyNic reagent vial Pipette the solution up and down to resuspend the reagent pellet Note before DMF addition there is a small but visible pellet at the bottom of the vial 2 Add 1 5 ul dissolved S HyNic reagent to the antibody solution 100 ul 1mg mL prepared in the previous section Pipette the solution up and down to mix 3 Incubate the reaction for 2 h at room temperature D Buffer Exchange IgG 3 minutes 1 Five minutes before the end of the HyNic modification reaction prepare a spin column yellow cap by twisting off the bottom closure and loosening the yellow cap do not remove Place the spin column into a collection tube provided 2 Mark the top of the yellow cap using an indelible pen to identify the sample Also place a vertical mark on the side of each spin column as shown on the next page Label the lid w sample ID Place pen mark on Side of spin column Collection tube 3 Place the assembly into the c
13. entrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor Use an appropriate balance tube opposite the assembly Ifi Catalog A 9001 006 12 sol U Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided Open the yellow cap load the now completed antibody HyNic modification reaction 100 uL to the top of the dry resin bed loosely cap and place the column back into the collection tube Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes Use a balance tube opposite the spin filter After centrifugation transfer the solution from the bottom of the collection tube to a 1 5 mL tube Label the sample appropriately e g HyNic IgG E Conjugate Formation 2 h 1 3 Spin the dark brown vial containing 4FB modified R PE 5 seconds 9 1000 x g to collect the contents at the bottom of the tube Transfer 48 ul 4FB modified R PE to the tube containing HyNic modified antibody pipette up and down to mix Incubate in the dark for 2 h at room temperature F Conjugate Purification Stage 1 Affinity Isolation 1 34 hours 1 Catalog A 9001 006 Magnetic Affinity Bead Prep Briefly centrifuge 100 uL affinity magnetic beads black slurry at 1000 x g for 5 seconds to col
14. le of Contents o kc Catalog A 9001 006 3 sol U 4 Chapter 1 Introduction A User Manual This manual provides instructions for using the R PE Antibody All in One Conjugation Kit This chapter contains the following sections Purpose of Manual Intended Users Customer Service and Technical Support B Purpose of Manual Each R PE Antibody All in One Conjugation Kit provides all the necessary reagents and components to produce two 2 R PE antibody conjugates in about 6 hours Use of the kit for each antibody results in e The modification of a user supplied antibody 100 ug with S HyNic e he conjugation of a HyNic modified antibody with 4FB PE resulting in the formation of an R PE Antibody conjugate e The isolation of highly purified R PE Antibody conjugates using a dual purification process magnetic affinity beads and Q spin filter C Intended Users The R PE Antibody All in One Kit is designed for users with minimal or no conjugation experience allowing them to prepare customized high purity ready to use R PE Antibody conjugates in 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 oolulink The Conjugation Company 9853 Pacific Heights Blvd Ste H san Diego CA 92121 it Catalog A 9001 006 4 sol U Ir Chapter 2 Overview of Conjugation
15. lect the bead contents at the bottom of each tube Place the beads on a magnet for 10 seconds to pellet Remove and discard the supernatant Add 400 uL Buffer B to the bead slurry and gently vortex to mix Before the beads resettle place the beads on the magnet for 10 seconds to pellet then carefully remove and discard the supernatant without disturbing the pellet solulink 4 Wash the beads by repeating steps 2 and 3 two 2 additional times After discarding the final wash immediately proceed to the next step Bind Conjugate to Affinity Beads 1 Away from the magnet add the completed conjugation reaction 150 uL from step E 3 to the washed bead pellet Gently mix the slurry up and down 3 4 times using a P 1000 pipette Never vortex the beads after the conjugate mixture is added Tip set the volume on the P 1000 pipetman on 100 uL before pipette mixing 2 Let the slurry settle and incubate for 10 minutes away from the magnet 3 Gently pipette the bead slurry up and down with a P 1000 pipette 3 4 times to remix and incubate for another 10 minutes away from the magnet 4 Repeat step 3 one 1 additional time total incubation time 30 minutes away from the magnet 5 Away from the magnet gently pipette the settled slurry up and down to mix and immediately place the tube on the magnet before the beads resettle Once the beads pellet to the side 10 20 seconds discard the supernatant without disturbing the
16. ly mix the antibody HyNic reaction mixture remove all non protein amine contaminants such as glycine or Tris before modification keep and store S HyNic sealed in the aluminum pouch containing dessicant measure the initial antibody concentration before proceeding Bradford or NanoDrop concentrate or dilute the antibody sample into the recommend range 1 mg ml and 10 ul before proceeding solulink Low conjugate and or low spin column recovery volume calibrated variable speed antibody recovery microcentrifuge to the indicated speed and time e g 1500 x g Lower or higher speeds can adversely compromise protein and volume recovery H References 1 Dirksen A Hackeng T Dawson P 2007 Nucleophilic Catalysis of Oxime and Hydrazone Reactions by Aniline ACS Poster 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 S Lim P Manusu A A Gooley L Williams and D B Rylatt Purification of monoclonal antibodies from ascitic fluid using preparative electrophoresis Journal of Chromatography A Vol 827 Issue 2 11 December 1998 Pages 329 335 5 Francesca Chiodi Dr Sid n Eva sby 2005 Isoelectric f
17. n cap do not remove Place the spin column into a collection tube provided Mark the top of the brown cap using an indelible pen to identify the sample Also place a vertical mark on the side of each spin column as shown below Label lid w conjugate ID Place pen mark on side of spin column lt Collection tube it Catalog 9001 006 15 solu Ir 3 Place the assembly into the centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor Use a balance tube opposite the spin filter 4 Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided 5 Open the brown cap load 150 uL eluted conjugate to the top of the dry resin bed loosely cap and place the column back into the collection tube 6 Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes Use an appropriate balance tube opposite the spin filter 7 After centrifugation add 300 uL Buffer E to 150 uL of conjugate at the bottom of the collection tube and pipette up and down to mix 450 pL total Set aside on the bench H Conjugate Purification Stage 2 Q Spin Filter 35 min 1 Pre wet a spin filter by adding 200 uL Buffer E to the top of the unit see below and incubate for 2 minutes filter unit y
18. nother 4 minutes at 2 000 x g The highly colored conjugate 150 uL is now located at the bottom of the collection tube Set the collection tube aside on the bench I Buffer Exchange Conjugate 3 minutes 1 Prepare a spin column blue cap by twisting off the bottom closure and loosening the brown cap do not remove Place the spin column into a collection tube provided 2 Mark the top of the blue cap using an indelible pen to identify the sample Also place a vertical mark on the side of each spin column as shown below it kc Catalog A 9001 006 17 sol U Iri Label lid w conjugate ID amp 1 NN Place pen mark on Side of spin column Collection tube 3 Place the assembly into the centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor Use an appropriate balance tube opposite the spin filter 4 Centrifuge at 1 500 x g for 1 minute Discard the flow through from the collection tube The column matrix will appear white in color Place the column back into a new empty collection tube provided 5 Remove the blue cap load 150 uL of the highly colored conjugate to the top of the dry resin bed loosely recap and place the column back into the collection tube 6 Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes Use a balance tube opposite the assembly 7 After centrifugation transfer
19. ntil the spectrum 220 350 nm appears in the window Note for precious or limited samples the 2 ul aliquot can be recovered from the pedestal o Catalog A 9001 006 22 sol U 9 Record the antibody concentration directly from the NanoDrop display window mg ml Alternately calculate the antibody concentration manually as illustrated in the following example on the next page Example A mouse IgG sample in PBS 100 ul 1 mg ml was scanned as described and its concentration confirmed in the sample below Figure 7 A mouse IgG sample 100 ul 9 1 mg ml in PBS pH 7 2 was scanned on the NanoDrop as described in the text Equation A so E1 x 10 mg ml protein concentration mg ml E196 mass extinction coefficient see Table 1 Mouse IgG 1 mg ml see Fig 7 Azgo reading from scan in Figure 6 1 34 Antibody E1 value Table 1 14 00 A280 E196 bovine IgG x 10 mg ml protein concentration mg ml 1 34 14 00 x 10 mg ml 0 96 mg ml Catalog A 9001 006 23 sol U Table 1 Mass extinction coefficients E196 used to determine IgG concentration E196 A280 Of 10 mg ml solution 1 cm path length D R PE Absorption Spectrum Unmodified R PE N H 100 lt Mex Absorbance ormalize Clot Abs Cor Sample ID 140 R PE 1 25 1 00 T4 0 75 0 50 Abs 1 0 00 0 10 1 t i i i i i 250 275 300 325 350 375 400 425 450 475 500 525 550 575
20. ocusing of monoclonal immunoglobulin G A and M followed by detection with the avidin biotin system Electrophoresis Vol 6 Issue 3 124 128 link Catalog A 9001 006 28 sol U Iri
21. or puncture the filter surface during this pipetting step 6 If necessary repeat steps 4 and 5 until the volume in the filter cup finally reaches 100 ul 1 mg ml 7 Transfer the concentrated IgG solution to a new 1 5 ml microfuge tube and proceed with the conjugation procedure F K Catalog A 9001 006 26 sol u Troubleshooting Guide Problem Poor conjugate yield Poor HyNic modification Poor HyNic modification Catalog A 9001 006 Possible Cause initial antibody concentration and volume were incorrect or unknown presence of protein carrier e g BSA or gelatin is contaminating the antibody sample improper mixing of HyNic reaction components major presences of amine contaminants with the antibody e g 80 mM Tris improper storage of S HyNic reagent can lead to hydrolysis of the NHS ester initial antibody concentration was too low or too high 27 Hecommended Action whenever possible verify the original protein concentration using a Bradford assay to assure 1 mg ml concentration concentrate or dilute the antibody sample to the required range 1 mg ml and 100 ul preservatives can interfere with the accuracy of a Bradford protein assay Remove all interfering preservatives such as thimerosal or proclin preservative remove and purify away all protein carriers such as BSA or gelatin using affinity chromatography before proceeding make sure to proper
22. pellet Wash Magnetic Beads 1 Add 400 uL Buffer C to the bead pellet and gently pipette P 1000 the slurry up and down several times to wash Allow the beads to resettle for 3 minutes on the bench top 2 Once again pipette the slurry up and down and immediately place on a magnet before the beads resettle Remove and discard the supernatant without disrupting the pellet 3 Repeat step 1 2 three 3 additional times always discard the supernatant between washes Ifi Catalog A 9001 006 14 sol U Iri Elute Conjugate from Beads 1 Hemove the beads away from the magnet and elute the conjugate by adding 50 uL Buffer D to the pellet Gently and slowly mix the bead slurry up and down with the aid of a pipetman Allow the beads to incubate away from the magnet for 4 minutes Hepeat step 2 and 3 two 2 additional times total incubation time 12 min Gently resuspend the settled slurry by pipetting up and down and immediately place the beads on the magnet before they resettle Transfer the highly colored supernatant 50 containing the eluted conjugate to a new 1 5 mL tube and label appropriately Continue elution of the conjugate by repeating steps 1 through 6 two 2 additional times pooling all conjugate fractions into the same tube 150 uL final volume G Buffer Exchange Conjugate 3 minutes 1 2 Prepare a spin column brown cap by twisting off the bottom closure and loosening the brow
23. rdous 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 9001 006 2 sol u 4 Chapter TE aid gels Ele 40 4 MEE duci 4 B Purpose ortiatllalasssiussteim 4 GE pe eB NR 4 D Customer Service and Technical Support eeeessseeeeeseeeeeeeen nennen eene nnne nnns 4 Chapter 2 Overview of Conjugation 5 MM O imm 5 B All in One Conjugation Technology nennen treten 5 C All in One Conjugation Process Summary 8 D Materials Provided and Storage 9 E Additional Materials Required But Not Provided cccccccssscccceessececeesececeesceeeeeseceseeecessegeceeseees
24. the purified conjugate solution 7150 pL from the bottom of the collection tube to a new 1 5 mL tube and label appropriately 8 Measure the final protein concentration of the conjugate using a Bradford or BCA protein assay see Appendix for reference pro Chapter 4 Appendix A Human IgG R PE Conjugate Monoclonal An example F Catalog A 9001 006 18 sol U 4 Coomassie stain UV Fluorescence 1 2 3 A4 5 1 2 3 4 5 1 1 OKT3 R PE n lt heterodimer 100 90 3 10 Denaturing SDS PAGE 20 1 MW Ladder 2 R PE 4FB modified M W 120 kD denatured 5 ug 3 mAb IgG 5 ug 4 Crude unpurified OKT3 R PE conjugate reaction 5 yg 5 All in One purified OKT3 R PE conjugate 5 ug Figure 5 SDS denaturing PAGE gel images Coomassie stained UV excitation of an All in One R PE antibody conjugate Note almost exclusive formation of the desired 1 1 heterodimer denatured M W 270 kD as well as the purity of the final conjugate lane 5 B Bradford Protein Assay Solulink highly recommends when IgG is not limiting or its concentration source or the quality is unknown that the antibody be assayed for initial protein concentration using a Bradford protein assay prior to using the All in One conjugation kit The quality and quantity of antibody are critical to the success of the procedure In this section of the solulink Catalog A 9001 006 19 appendix we pro
25. tion on a spectrophotometer proceed as follows solulink Catalog A 9001 006 21 1 Turn on the spectrophotometer and click on the NanoDrop icon to launch the software Place a 2 ul drop of molecular grade water on the clean pedestal click OK 3 When the main menu appears select the A239 menu option Note do not use the UV VIS menu option on the NanoDrop to read an antibody sample 4 After the Argo menu appears click off the 340 nm normalization option using the mouse Note some NanoDrop instruments do not have this feature and therefore can be ignored on these instruments 5 Inthe window labeled Sample Type select Other protein E196 option from the pull down menu Enter the appropriate E196 value from Table 1 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 a flat baseline 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 Transfer 2 ul antibody to the pedestal and click the Measure icon Wait u
26. ugate residual 4FB PE residual HyNic IgG C V ee Bind and elute conjugate from affinity magnetic beads Purify Conjugate a Buffer exchange 1 h 45 min 1 w brown cap spin column il Bind and elute conjugate to Q spin filter 1 Buffer exchange wiblue cap spin column R PE IgG conjugate 1 1 heterodimer Figure 4 All in One conjugation process summary k Catalog A 9001 006 8 sol u 4 D Materials Provided and Storage Conditions Components Amount Storage conditions S HyNic 2 100 ug Keep desiccated at or below room temperature within desiccated foil pouch Buffer F Keep refrigerated 2 8 C Anhydrous DMF 500 uL Keep refrigerated 2 8 C Affinity Magnetic Beads 2x100uL Keep refrigerated 2 8 C Red Cap Spin Column Keep refrigerated 2 8 C 2 Q Spin Column amp Q 2 4 Room temperature or refrigerated Collection Tubes 2 8 C Collection Tubes 16 Room temperature 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 Microcentrifuge e g Eppendorf or MicroMax 1 5 ml microfuge tubes Chapter 3 All in One Conjugation Protocol If kc Catalog A 9001 006 9 solu Ir Note Before use remove the kit from the refrigerator and allow all kit components to warm up to room temperature for at least 30 minutes A Ig
27. vide a reference protocol for measuring antibody or conjugate protein concentration with Bradford protein assay reagents not provided Bradford Plate 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 4 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 Prepare the following protein dilution standards and blank as follows Add 80 ul 2 mg ml bovine IgG standard to 120 ul PBS 0 8 mg ml standard Add 75 ul 0 8 mg ml standard to 25 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 Pipette 5 ul of each standard and blank along with duplicates of the antibody sample to plate wells Add 100 ul of previously diluted dye reagent 1 4 to each well and mix thoroughly Always replace pipette tips between additions Incubate at room temperature for 5 10 minutes but no more than 60 minutes Measure absorbance at 595 nm on a suitable plate reader F Catalog A 9001 006 20 sol U 7 typi
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