Antibody-Oligonucleotide All-in-One Conjugation Kit User Manual
Contents
1. 4 Measure the A260 of a 2 uL aliquot of the 1 1000 4FB oligo dilution as displayed in the 10 mm path length window 5 Enter the resulting A260 into Conjugation Calculator Section D green cell The calculator will then display the concentration of the 4FB oligo in units of A260 uL Section D 1 yellow cell If the calculator displays YES Section D 2 yellow cell then proceed to step 6 below If the calculator displays FALSE Section D 2 yellow cell proceed to step 7 below 6 When the measured 4FB oligo concentration is in the required range 0 3 to 0 6 OD260 uL proceed to measure the oligo 4FB Molar Substitution Ratio as described in Section of this protocol Leave the 4FB Oligo solution in the concentrator unit until after Section is complete 7 If the 4FB oligo concentration displayed is greater than 0 6 OD260 uL dilute the 4FB oligo in the filter unit by adding the solulink www Solulink com 19 Antibody Oligonucleotide All in One Conjugation Kit indicated volume of Solution C uL from the Conjugation Calculator Section D 3 yellow cell to obtain 0 6 OD260 uL Then re enter the adjusted value 0 6 OD260 uL into the Conjugation Calculator Section D green cell Once the 4FB oligo is adjusted to 0 6 OD260 uL proceed to measure the oligo 4FB Molar Substitution Ratio as described in Section of this protocol Leave the 4FB Oligo solution in the concentrator unit until after Section is com2. 100 uL PBS buffer blank 2 In a flat bottom 96 well plate prepare standards by pipetting 10 uL of each standard and a blank into 100 uL Bradford working solution mix Replace pipette tips between additions 3 In an adjacent well containing 100 uL Bradford working solution add 10 uL of the conjugate 4 Incubate at room temperature 18 25 C for 15 min do not exceed 60 min 5 Measure absorbance at 595 nm using pre programmed Bradford assay software 6 Data from a typical Bradford assay is provided as an illustration only in Figure 6 solulink www Solulink com 32 Antibody Oligonucleotide All in One M Conjugation Kit File Edit View a Control Assays Graph Window Help ane w aan Bradford Protein Assay Plated SM Setup E Template E Template E Reduction E Display BE Display A o 2 TT TT Atomis Once Calibrate On Column Priority Plate Last Read 3 32 Pd 101302009 NN TN TN NA TN DN DN DN Wavelength Combination Lir Data Mode Absorbance Plate Blank Used Lind 0 362 jii Standards pole ra TN Unknowns uH E4 Unknowns OD Values Std Dev Adj Cone 0 197 e ir BEEE 2z fe owsa os oss oo oo 1o 0183 iste omo omso ooo oo to ono FEE Unkno diln PJE wv L Standard Curvelit Fit Standard Curve Mean OD Yalue 0 26 Concentration yo A Ox A o 37081 Standards Concentration vs Mean OD val Figure 6 Bradford output from a comme
3. 10 uL Conjugate Quench Reagent and incubate for 10 minutes at room temperature B Conjugate Purification 1 Centrifuge the vial containing affinity magnetic beads black slurry at 1000 x g for 5 seconds to collect the bead contents at the bottom of the tube 2 Add 500 uL Solution C to the bead slurry using a P 1000 pipette pipette the solution up and down several times to mix the slurry Quickly before the beads resettle place the tube on the magnet for 10 seconds carefully remove and discard the supernatant using a P 200 pipette without disturbing the pellet 3 Repeat step 2 three 3 additional times to fully wash the beads removing the supernatant after each wash 4 Immediately add the quenched conjugation reaction 115 uL directly to the washed bead pellet solulink www Solulink com 29 Antibody Oligonucleotide All in One Conjugation Kit 5 Gently pipette the slurry conjugate mix up and down 3 4 times with a P 1000 barrier tips Set a timer and allow the settled slurry to incubate for 10 minutes away from a magnet Never vortex beads after conjugate addition Set P 1000 to 90 uL when mixing slurry 6 Repeat step five three 3 additional times for a total conjugate binding time of 40 minutes Some minor but unavoidable bead loss can occur due to non specific binding of beads inside the pipette tip 7 Gently pipette the settled slurry up and down one last time and immediately place the slurry on the magnet
4. B All in One M Conjugation Technology oooooooooooooooo 4 C All in One M Conjugation Process oooooooooooomm 6 D Starting Antibody Requirements W Woo WWW mmm 10 E Starting Amino Oligonucleotide Requirement oooooWoo 10 ig I Component Rn RA Aoa 11 G Materials to be Provided by the User co Wo o Woo 11 H Component Storage Conditions ooWo oo Wa 11 Chapter 3 Conjugation asa 12 Stage 1 Modification of Amino Oligonucleotide with Sulfo S 4FB 12 A Enter Amino Oligo Information into Conjugation Calculator 12 5 Me SUS NG AMIN DIGO mein eren aan ia oa Na ai 13 C Measure Amino Oligo Concentration on a Spectrophotometer 14 D B fer Exchange AMINO Old Ooo ekes saka sae samaan mtma 15 E Dissolve Sulfo S 4FB Reagent ooo ooW Wo Woo WWW ma 16 F Modify Amino Oligo with Sulfo S 4FB Reagent oooooo oo 16 G Buffer Exchange and Concentrate 4FB Oligo oooooWoWo 17 H Measure 4FB Oligo Concentration oo ooooW oom WWW 19 Measure and Quantify 4FB Molar Substitution Ratio 21 Stage 2 Modification of Antibody with S HyNic eee 24 A Antibody PreparafiOi o o o oo oo oo oWoomoo ooo ieeanbnasnaa 24 B Confirm Antibody Concentration on a Spectrophotometer 25 C Buffer Exchange Antibody cccccceecc
5. Desiccated Sulfo S 4FB Reagent Desiccated solulink www Solulink com 11 Antibody Oligonucleotide All in One Conjugation Kit Chapter 3 Conjugation Protocol Prior to Starting The conjugation protocol is a three stage process 10 5 hours in duration where each step takes several hours to complete If desirable the end user can complete Stage 1 on the first day 4 hr then proceed with Stages 2 and 3 on day two 6 5 hr Keep in mind that we do not recommend stopping the procedure after Stage 2 The only convenient stopping point throughout the entire protocol is immediately after Stage 1 so we recommend that you schedule and plan your time accordingly Total hands on is approximately 4 hrs If the starting amino oligo is in dry pellet form If the amino oligo to be modified with Sulfo S 4FB is in dry pellet form and contains a minimum of 10 OD260 units and no more than 40 OD260 units proceed to Stage 1 If more than 40 OD260 units are provided by the vendor in a dry pellet form do not make adjustments to the OD260 units at this time and proceed to Stage 1 If the starting amino oligo is in a liguid form If the amino oligonucleotide to be modified with Sulfo S 4FB is already in liguid form and its concentration is known units of OD260 uL then transfer to another tube a volume eguivalent to a minimum of 10 OD260 units and no more than 40 OD260 units and concentrate into a dry pellet form using a vacuum concentrator e g
6. O Check the baseline and reblank if necessary to insure that it is flat d Measure the A260 of the 1 250 amino oligo as displayed in the 10 mm path length window Record the A260 value 2 Enter the recorded A260 into the Conjugation Calculator Section B green cell The calculator determines the A260 uL as well as the total OD260 oligo units available for conjugation Section B yellow cells A minimum of 10 OD260 and a maximum of 40 OD260 units are required Important If less than 10 OD260 units are recovered after resuspension obtain additional amino oligo If greater than 40 OD260 are resupended transfer an aliquot equivalent to 40 OD260 units into another tube and bring the final volume to 100 uL with Solution A then proceed with the protocol solulink www Solulink com 14 Antibody Oligonucleotide All in One Conjugation Kit Conventional UV VIS Spectrophotometer 1 Determine the concentration OD260 uL of the resuspended amino oligo using a quartz micro cuvette 50 100 uL 1 cm path length and a spectrophotometer as follows remember to use barrier tips a Ina41 5 mL tube prepare a 1 250 dilution of the resuspended amino oligo by transferring 1 uL with a calibrated P 2 pipette into 249 uL molecular grade H20 b Blank the spectrophotometer at 260 nm using molecular grade H O c Measure the A260 of the 1 250 amino oligo Record the A260 value 2 Enter the recorded A260 into the Conjugation Calculator Secti
7. Place the column back into a new empty collection tube provided 5 Apply the dissolved amino oligo 10 40 OD260 units per 100 uL in Solution A to the top of the dry resin bed Place the spin column into the empty collection tube Loosely recap and properly orient the spin column in the centrifuge Centrifuge at 1 500 x g for 2 min Important Rotor speed must be set to 1500 x g RCF and not 1500 x rom RPM The volume of oligo recovered in the collection tube should always be approximately the same volume that is loaded on the spin column For example when 100 uL of amino oligo is loaded 100 10 uL should be recovered If the recovered volume is low it is likely that rotor speed is not calibrated If this happens re centrifuge the spin column at 1 500 x g speed for an additional minute to recover any trapped solution the spin column 6 Measure the recovered volume uL of amino oligo at the bottom of the collection tube using a P 200 pipette and transfer it to a new 1 5 mL tube Note Yield in A260 units through a spin column is generally gt 90 for amino oligos ranging in size from 35 60 bases Recovery yields from smaller oligos e g 20 mers are somewhat lower e g 75 due to the size exclusion limit of the spin column matrix Never spin oligos smaller than 20 mers through a spin column to avoid oligo loss 7 Label the tube with the corresponding oligo ID and volume uL recovered The amino oligo is now ready for 4FB modifica
8. i e no offsets Clean the pedestal and measure the A280 of the antibody sample with a 2 uL aliquot of antibody sample Record the A280 Enter the name of the antibody the measured A280 10 mm path length and the total volume of antibody solution into the Conjugation Calculator Section F green cells The calculator displays the protein concentration mg mL and the total mass of antibody to be conjugated into the Conjugation Calculator Section F yellow cells A concentration of 1 0 2 mg mL is required to proceed otherwise obtain additional IgG or adjust the concentration to 1 mg mL Note the calculator uses the average known mass extinction coefficient E1 of IgG to calculate protein concentration e g E1 14 Antibody Concentration on a Conventional Spectrophotometer 1 www Solulink com 26 Blank the spectrophotometer at 280 nm using an appropriate blank solution e g the solution used to resuspend the antibody with a quartz micro cuvette 50 100 uL 1 cm path length Empty the cuveite solulink Antibody Oligonucleotide All in One Conjugation Kit 2 Measure the A280 of the antibody sample Record the A280 and recover the antibody sample from the cuvette back to its sample tube 3 Enter the name of the antibody the A280 1 cm path length and the volume of antibody solution e g 100 uL into the Conjugation Calculator Section F green cells The calculator then displays the protein co
9. inside the concentrator unit at this time and proceed to measure the 4FB oligo concentration on a spectrophotometer Note If the filter is not sufficiently or properly rinsed some 4FB oligo can remain bound to the filter surface Leave the 4FB oligo solution in the filter unit until the 4FB oligo concentration OD260 uL is confirmed on the spectrophotometer H Measure 4FB Oligo Concentration Measure the concentration of 4FB modified oligonucleotide OD260 uL within the filter concentrator body using a micro volume UV VIS scanning spectrophotometer e g NanoDrop ND 1000 or a conventional spectrophotometer When using a conventional spectrophotometer a quartz micro cuvette 50 100 uL 1 cm path length is required Use the instructions below depending on the specific type of spectrophotometer available to you NanoDrop or Conventional Concentration Using a Micro Volume NanoDrop Spectrophotometer 1 Prepare a 1 1000 dilution of the 4FB modified oligo by transferring 1 uL calibrated P 2 pipette from inside the spin filter concentrator body to a 1 5 mL tube containing 999 uL molecular grade H20 Label the tube with the appropriate oligo ID 2 Select the Nucleic Acid menu option on the NanoDrop and initialize the instrument 3 Clean the sample pedestal and blank the instrument with molecular grade H20 Confirm a flat baseline by clicking on the Re blank icon and reblank if necessary Clean the sample pedestal dry
10. not available use a rapid and brisk downward flick of the sample vial in an attempt to collect as much of any adhering liquid at the bottom of the vial B Confirm Antibody Concentration on a Spectrophotometer Confirm the resuspended antibody concentration by measuring the sample s A280 on a spectrophotometer As before either a micro volume UV VIS scanning spectrophotometer e g NanoDrop ND solulink www Solulink com 25 Antibody Oligonucleotide All in One Conjugation Kit 1000 or conventional spectrophotometer can be used When using a conventional spectrophotometer a quartz micro cuvette 50 100 uL 1 cm path length is required Use the appropriate instructions that follow depending on the specific type of spectrophotometer available to you NanoDrop or Conventional Antibody Concentration on a NanoDrop Spectrophotometer 1 2 Launch the NanoDrop software by clicking the desktop icon Select the A280 menu option Initialize the instrument with 2 uL molecular grade water on a clean pedestal When the scan window appears turn off the 340 nm normalization feature by clicking the appropriate box Note some NanoDrop V instruments do not have a 340 nm normalization feature and ignored for those instruments Blank the spectrophotometer using 2 uL of the appropriate buffer blank solution e g the solution used to resuspend the antibody Click the Reblank icon to verify a flat baseline
11. of the rotor Use an appropriate balance tube opposite the spin column 5 Centrifuge at 1 500 x g for 1 minute Discard the flow through from the bottom of the collection tube The column matrix will solulink www Solulink com 28 Antibody Oligonucleotide All in One Conjugation Kit appear white in color Place the column back into a new empty collection tube provided 6 After the HyNic modification reaction is complete apply the HyNic IgG reaction mixture 100 uL to the top of the dry resin bed Loosely recap and orient the spin column in the centrifuge Centrifuge at 1 500 x g for 2 minutes 7 Transfer the recovered volume uL of HyNic modified IgG using a P 200 pipette from the bottom of the collection tube to a new 1 5 mL tube Measure and record the volume recovered and immediately proceed to conjugate formation Stage 3 Formation of Conjugate and Purification A Conjugate Formation 1 Enter the name of the antibody the name of the 4FB oligonucleotide and the volume of HyNic lgG to be conjugated into the Conjugation Calculator Section G green cells 2 Add the indicated volume uL of 4FB modified oligonucleotide displayed in the Conjugation Calculator Section G yellow cell to the HyNic modified antibody Pipette the solution up and down to mix 3 Incubate the antibody oligo conjugation reaction for 2 hr at room temperature 4 At the end of the conjugation reaction quench the reaction by adding
12. or has been damaged N Repeat steps 2 through 6 four 4 additional times to completely buffer exchange and concentrate the 4FB oligo Do not skip any of the spin steps Important Although five spin cycles are time consuming and tedious total time 1 h proper execution of this step is critical to the success of the conjugation reaction by removing excess Sulfo S 4FB Oo After the final spin check the volume in the concentrator unit If the final volume is less than 25 uL simply adjust the volume to 25 uL by adding a small aliquot of Solution C If the final volume is greater than 25 uL continue to centrifuge the spin filter for a few more minutes until the volume reaches 25 uL co Open the lid of the filter unit and using a P 20 pipette carefully pipette the solution up and down 15 times to fully resuspend the 4FB oligo 10 Using the same pipette rinse the filters surface 5 or 6 times with the oligo solution by repeatedly pipetting the 4FB oligo solution over the entire surface of the filter This rinsing process insures that any filter bound 4FB oligo is brought back into solution 11 Close the lid of the filter unit and insert it back into the collection tube Briefly centrifuge for 10 seconds at 1 000 x g to collect the full 25 uL of 4FB oligo back at the bottom of the concentrator unit solulink www Solulink com 18 Antibody Oligonucleotide All in One Conjugation Kit 12 Leave the 4FB modified oligo
13. the concentrator body from the collection tube and with a rapid inverted flick of the wrist discard the entire volume of Solution C from the concentrator body into a suitable waste receptacle Place the empty concentrator body back into the collection tube Buffer Exchange 4FB Oligo 1 Transfer the completed Sulfo S 4FB amino oligo modification reaction into the empty concentrator body 100 125 uL 2 Add 400 uL Solution C to the concentrator body to bring the total volume to approximately 500 uL solulink www Solulink com 17 Antibody Oligonucleotide All in One Conjugation Kit 3 Using a P 1000 pipette mix the solution in the concentrator body up and down with pipette action 10 15 times without touching or damaging the filter surface 4 Close the lid and mark the filter unit with an identifying name or ID 5 Orient the oligo spin filter in the centrifuge so the volume calibration numbers face toward the center of the rotor Remember to use an appropriate balance tube opposite the oligo spin filter unit 6 Centrifuge at 15 000 x g for 12 min After centrifugation the volume in the concentrator body will generally be between 25 and 50 uL some translucent color may be associated the concentrated solution e g light brown Note We recommend as a precautionary measure after the first spin that you may wish to retain the flow through from the bottom of the collection tube just in case the filter membrane is defective
14. 00 x g for 10 seconds using a larger tabletop centrifuge If a larger tabletop centrifuge is not available use a rapid and brisk downward flick of the sample vial in an attempt to collect as much of any adhering liquid at the bottom of the vial If the IgG is already in liquid form 1 If the initial antibody sample is already in liquid form at 1 mg ml transfer 100 ul to another labeled tube 100 ug If the initial antibody sample is in liquid form at a concentration greater than 1 mg ml transfer a volume equivalent to 100 ug to another tube and add the necessary volume uL of Solution B to obtain 100 uL at 1 mg ml And finally if the initial antibody sample is less than 1 mg ml the sample must first be concentrated to 1 mg mL and 100 uL using a Suitable ultra filtration spin filter Spin filters are available from various vendors e g Amicon or Sartorius An ultra filtration spin filter is not provided with this kit 2 Briefly centrifuge the resuspended antibody at 1 000 x g for 10 seconds to collect the entire liquid contents at the bottom of the Original vial and proceed to confirm antibody concentration Note if the original IgG product is packaged in a product vial that is too large to fit inside a standard microcentrifuge Such larger vials e g glass vials can first be placed inside a 50 mL disposable conical tube and briefly soun at 1000 x g for 10 seconds using a larger tabletop centrifuge If a larger tabletop centrifuge is
15. 1 11 If 4FB MSR of the oligo is determined to be in the acceptable range e g greater than 0 5 and less than 1 1 transfer the 4FB solulink www Solulink com 23 Antibody Oligonucleotide All in One Conjugation Kit oligonucleotide still in the concentrator unit to a new 1 5 mL tube Label the tube with the MSR and the OD260 uL and store at 4 C for 1 month or up to 1 year at 20 C This is the end of Stage 1 anda convenient stopping place Note An ACCEPTABLE MSR is displayed if the oligo is at least 50 4FB modified i e MSR 0 5 MSR values lower than 50 can occur for various reasons including the absence of the amino group or insufficient purity of the oligo Do not proceed if the calculated 4FB MSR is lower than 0 5 A value greater than 1 0 is occasionally observed and is usually the result of incomplete desalting slight excess of Sulfo S 4FB carryover Values up to 1 1 are acceptable but an additional desalting concentration cycle as previously described Section G are recommended when values greater than 1 1 are observed Stage 2 Modification of Antibody with S HyNic Antibodies are packaged in two different physical forms solids and liquids Individual samples can vary greatly from vendor to vendor and are often sold in a variety of different sizes and or concentrations In all cases Solulink highly recommends starting with the highest quality purity antibody available Depending on the initial form so
16. 60 nucleotides in length to any monoclonal or polyclonal IgG class antibody The protocol requires a minimum quantity of 10 ODzg and a maximum of 40 ODseo units of HPLC purified amino oligonucleotide Solulink recommends that longer oligo sequences e g gt 49 mer be synthesized with a 5 amino group and shorter oligos lt 49 mer with a 3 amino group if the specific application permits Oligonucleotides lt 49 mer can be either reverse phase RP or ion exchange purified IEX while longer oligos gt 49 mer can be IEX or double HPLC purified depending on the specific services offered by each vendor Some vendors offer these purification options on a custom basis while others offer them as a standard service albeit at additional cost Be advised that unpurified 3 amino oligos contain a significant quantity of truncated failure sequences that lead to undesirable conjugation products while unpurified 5 amino oligos contain up to 50 of A260 units in the form of shorter unmodified failure sequences that never form conjugate and thereby alter the stoichiometry of the conjugation reaction For best results always use the highest quality HPLC purified amino oligonucleotide available Note Please be advised that some oligo vendors will not HPLC purify amino modified oligos or in some cases longer oligonucleotide sequences modified or unmodified except as a custom service However some oligo suppliers do offer these services as a Standard optio
17. Antibody Oligonucleotide All in One M Kit V 06 18 10 solulink Antibody Oligonucleotide All in One Conjugation Kit User Manual Catalog No A 9202 001 www solulink com The products offered here are for research use only Any commercial application will reguire 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 No license is granted or implied to any patents to technologies for which the end user applies our products 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 2009 Solulink The Conjugation Company 9853 Pacific Heights Blvd
18. Do not proceed if the measured 4FB ratio is outside the required range 12 If 4FB MSR of the oligo is determined to be in the acceptable range e g greater than 0 5 and less than 1 1 transfer the 4FB oligonucleotide still in the concentrator unit to a new 1 5 mL tube Label the tube with the MSR and the OD260 uL and store at 4 C for 1 month or up to 1 year at 20 C This is the end of Stage 1 anda convenient stopping place Note An ACCEPTABLE MSP is displayed if the oligo is at least 50 4FB modified i e MSR 0 5 MSR values lower than 50 can occur for various reasons including the absence of the amino group or insufficient purity of the oligo Do not proceed if the calculated 4FB MSR is lower than 0 5 A value greater than 1 0 is occasionally observed and is usually the result of incomplete desalting slight excess of Sulfo S 4FB carryover Values up to 1 1 are acceptable but an additional desalting concentration cycle as solulink www Solulink com 22 Antibody Oligonucleotide All in One Conjugation Kit previously described Section G is recommended when values greater than 1 1 are observed 4FB Molar Substitution Assay Conventional Spectrophotometer 1 Prepare the 2 HP blank solution by adding 2 uL Solution C to 18 uL 2 HP Reagent label 2 HP Blank 2 Prepare a 4FB oligo sample by adding 2 uL 4FB modified oligo 0 3 0 6 OD260 uL to 18 uL 2 HP reagent label 4FB Oligo 3 Incubate the 2 HP
19. SpeedVac M from Savant Instruments then proceed to Stage 1 If the initial amino oligo to be modified is already in liquid form and its concentration is unknown units of OD260 uL then measure its concentration as described in this Chapter Section C Transfer into another tube a volume equivalent to a minimum of 10 OD260 units and no more than 40 OD260 units and concentrate into a dry pellet form using a vacuum concentrator then proceed to Stage 1 Stage 1 Modification of Amino Oligonucleotide with Sulfo S 4FB A Enter Amino Oligo Information into Conjugation Calculator 1 Enter the following amino oligo parameters directly from the Oligo vendor s Certificate of Analysis into the Conjugation Calculator Section A green cells a Oligonucleotide name as listed on the Certificate of Analysis b Total OD260 units as listed on the Certificate of Analysis solulink www Solulink com 12 Antibody Oligonucleotide All in One Conjugation Kit c Oligonucleotide molar extinction coefficient liter cm mol as listed on the Certificate of Analysis d Oligonucleotide molecular weight Daltons as listed on the Certificate of Analysis e Nanomoles of amino oligonucleotide as listed by vendor on Certificate of Analysis Important Enter the total OD260 units and the number of nanomoles provided by the vendor on the Certificate of Analysis even if only a portion of the total OD260 units provided are going to be modified The calculator requ
20. Suite H San Diego California 92121 All trademarks trade names service marks or logos referenced herein belong to their respective companies For research purposes only Not for diagnostic use www solulink com i Safety Information WARNING 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 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 www Solulink com iI Antibody Oligonucleotide All in One M Conjugation Kit Table of Contents ne en ne enna ene E ene een ee ene ater eee eee 2 Pes US OMA Udah ag la taat metana mahal E ion 2 B Purpose of ManUual WWoWo Wo W WWW mna 2 G Intended User eko enek ee Re noer 2 D Customer Service and Technical SUppOFt ooWooW Woo 2 Chapter 2 Overview of Conjugation sxciccee cecccsb mo esa memanas 3 A Product Description sobs abet aa Aan SN AA AA TA 4
21. blank and 4FB Oligo reactions at 37 C for 30 minutes 4 Prepare a 1 10 dilution of the 2 HP blank by transferring 10 uL from the completed reaction mixture into 90 uL molecular grade H20 then prepare a 1 100 dilution of the 2 HP blank by transferring 10 uL from the 1 10 2 HP blank dilution into a second tube containing 90 uL molecular grade H20 Label both tubes appropriately 5 Prepare a 1 10 dilution of the 4FB oligo by transferring 10 uL from the completed reaction mixture into 90 uL molecular grade H O then prepare a 1 100 dilution of the 4FB oligo by transferring 10 uL from the 1 10 4FB oligo dilution into a second tube containing 90 uL molecular grade H O Label both tubes appropriately 6 In a quartz micro cuvette blank the spectrophotometer with 90 uL 1 10 2 HP blank at 360 nm Remove the blank solution from the cuvette 7 Measure the A360 of the 1 10 4FB Oligo sample in the cuveite Record the A360 Clean the cuvette 8 Reblank the spectrophotometer using the 1 100 2 HP blank at 260 nm Remove the blank solution from the cuvette 9 Measure the A260 of the 1 100 4FB oligo sample Record the A260 Clean the cuvette 10 Enter the resulting A360 and A260 values into the Conjugation Calculator Section E green cells The calculator then displays the 4FB molar substitution ratio or MSR Section E yellow cell The calculator will display a warning if the 4FB MSR is too low or too high e g less than 0 5 and greater than 1
22. cleotide conjugate e The affinity purification of an antibody oligonucleotide conjugate that is stable for up to 1 year when stored at 4 C C Intended Users The Antibody Oligonucleotide All in One Kit is designed for users with minimal or no conjugation experience allowing them to prepare a single customized high purity ready to use antibody oligonucleotide conjugate 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 Solulink The Conjugation Company 9853 Pacific Heights Blvd Ste H San Diego CA 92121 solulink www Solulink com 2 Antibody Oligonucleotide All in One M Conjugation Kit Chapter 2 Overview of Conjugation This chapter provides An introduction to the Antibody Oligonucleotide All in One M Conjugation Kit An overview of the bioconjugation technology used to prepare antibody oligonucleotide conjugates Starting antibody and oligonucleotide requirements A list of required components and those to be provided by the user along with storage conditions This chapter contains the following sections A I D mM O O DW Product Description All in One Conjugation Technology All in One M Conjugation Process Starting Antibody Requirements Starting Oligonucleotide Requirements Kit Components Materials Provided by the User C
23. d conjugate is now in storage buffer at the bottom of the two collection tubes Pool the two 100 uL fractions containing antibody oligo conjugate into a single 1 5 mL tube Label and store the tube at 4 C for up to 1 year 6 Measure the protein concentration of the conjugate using a Bradford protein assay as described in the Appendix solulink www Solulink com 31 Antibody Oligonucleotide All in One Conjugation Kit Chapter 4 Appendix A Bradford Protein Assay A Bradford or BCA Protein Assay is used to determine the final antibody oligonucleotide conjugate concentration A reference protocol is provided for each procedure Bradford 96 Well Procedure Required Materials Bradford Reagent Bio Rad Hercules CA Cat 500 0006 96 well plate polystyrene flat bottom PBS Phosphate Buffered Saline P 200 and P 1000 pipettes and sterile tips Bovine IgG Antibody Standard 2 mg ml Pierce ThermoFisher Cat 23212 Molecular grade water Assay Protocol 1 Prepare 2 mL of Bradford working solution by adding 400 uL Bradford dye reagent to 1600 uL molecular grade water 1 4 ratios Prepare IgG standards and a blank in 1 5 mL tubes as follows Add 100 uL 2 mg mL bovine IgG standard to 300 uL PBS 0 5 mg mL standard Add 200 uL 0 5 mg ml standard to 200 uL PBS 0 25 mg mL standard Add 200 uL 0 25 mg mL standard to 200 uL PBS 0 125 mg mL standard Add 200 uL 0 125 mg mL standard to 200 uL PBS 0 0 0625 mg mL standard
24. de functional group formylbenzamide 4FB at the desired terminus of the oligonucleotide IgG g AR A A 3 or 5 amino modified oligonucleotide FF Xi S HyNic Sulfo S 4FB Ws N gt 4 2 4FB oligo HyNiclgG i RN w ee O O p i Q i Zi Ni pa Ne aS Conjugate ka Aniline catalyst Figure 1 Reaction of HyNic modified IgG with 4FB modified oligo leads to the rapid formation of a stable antibody oligonucleotide conjugate solulink www Solulink com 4 Antibody Oligonucleotide All in One M Conjugation Kit In a second stage of the process a polyclonal or monoclonal antibody 100 ug is modified using another HydraLink linker called S HyNic This NHS ester reacts with lysine residues incorporating HyNic functional groups hydrazinonicotinamide onto the antibody In the third and final stage the two modified biomolecules are mixed together in the presence of a reaction catalyst i e aniline to form the conjugate after which purification is carried out using a magnetic affinity solid phase 2 Conjugate Purification Antibody oligonucleotide conjugates produced with the All in One kit are ready to be used in the most demanding and sensitive downstream applications The kit delivers high purity conjugate virtually free of residual antibody or oligonucleotide gt 98 Reaction conditions are optimized to convert nearly 100 of the antibody into conjugate leaving only free excess 4FB ol
25. e All in One M Conjugation Kit Fah P HyNic HyNic E HAN sia x a Conjugate 4FB oligo to HyNic IgG STAGE 3 conjugate ag sii il aB MAN excess 4FB oligo NING aB MAN f ee 2 Bind conjugate to affinity magnetic beads ay Ta yo y E gt Wash away excess oligo on magnet Elute conjugate amp buffer exchange Nag ag P Figure 5 Stage Three 3 of the All in One process illustrates both the formation and purification of the conjugate solulink www Solulink com 9 Antibody Oligonucleotide All in One Conjugation Kit D Starting Antibody Requirements The Antibody Oligonucleotide All in One Conjugation Kit is designed to produce one 1 antibody oligonucleotide conjugate starting with 100 ug of any mammalian antibody regardless of IgG subclass and one amino modified oligonucleotide 10 40 OD260 units The quality and quantity of both the Starting antibody and oligonucleotide are critical to the success of the conjugation protocol We recommend using only the highest quality antibodies and oligonucleotides from trusted sources and reputable vendors This kit is not compatible with commercial antibody preparation containing added BSA or gelatin stabilizers If present these additives must be removed before proceeding E Starting Amino Oligonucleotide Requirements The Antibody Oligonucleotide All in One kit is designed to conjugate any high purity 5 or 3 amino modified oligonucleotide 20
26. eceeeeceeeeeeeeeesseeeesseeeesseeeeseeeeesaees 27 D Dissolve S AY NIC REAgeNnt soon mesh assets asah 28 E Modify IgG with S HyNic Reagent and Buffer Exchange o 28 Stage 3 Formation of Conjugate and Purification 29 Pe COMMU GALS FOU VO eect ses cece seca ees sce ecole cease OS 29 Be Gonjugate PUN CANON aa Moon an ana 29 C Buffer Exchange into Storage Buffer ooo oo Wo 31 Gio aon 6 aa E AS ene 32 A Bradford Protein Assay oooWo o Woo 32 B Antibody Oligonucleotide Conjugates Some Examples oo 34 C Troubleshooting Guide o Woo Wa 35 solulink www Solulink com 1 Antibody Oligonucleotide All in One Conjugation Kit Chapter 1 Introduction A User Manual This manual provides instructions for using the Antibody Oligonucleotide All in One M Conjugation Kit This chapter contains the following sections Purpose of Manual Intended Users Customer Service and Technical Support B Purpose of Manual Each Antibody Oligonucleotide All in One Kit provides all the necessary reagents and components to produce one 1 antibody oligonucleotide conjugate Use of the kit results in e The modification of one user supplied antibody and one amino oligonucleotide with HyNic and 4 FB moieties respectively e The conjugation of HyNic modified antibody with 4FB oligonucleotide resulting in the formation of an antibody oligonu
27. esin 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 7 Transfer the eluate from the bottom of the collection tube to a new labeled 1 5 mL tube measure the volume uL recovered from the collection tube with a P 200 pipette Label the tube with the appropriate volume uL recovered D Dissolve S HyNic Reagent 1 Add 35 uL DMF to a vial of S HyNic reagent Pipette the solution up and down for 60 seconds to dissolve the pellet E Modify IgG with S HyNic Reagent and Buffer Exchange 1 Add 2 0 uL of dissolved S HyNic modification reagent to the antibody sample Gently pipette the solution to mix 2 Incubate the antibody HyNic modification reaction at room temperature for 2 hours 3 Exactly five minutes prior to the end of the HyNic modification reaction prepare a spin column yellow cap by twisting off the bottom closure and loosening the cap do not remove Place the spin column into a collection tube and mark the top of the yellow cap with an indelible pen to identify the antibody sample Also place a vertical mark on the side of the spin column as shown on below Label lid w oligo ID l Ee Place pen mark on Side of spin column lt Collection tube 4 Place the assembly into the centrifuge and balance appropriately Orient the mark on the side of the spin column aiming outward and away from the center
28. exchange on spin column 3 Modify antibody with S HyNic 4 Bufferexchange on spin column 1 Conjugate 4FB labeled oligo to HyNic labeled IgG 2 Affinity purify conjugate 4FB MSR is an acronym for 4 formylbenzamide molar substitution ratio solulink www Solulink com 6 Antibody Oligonucleotide All in One M Conjugation Kit HN AN Resuspend amino oligo ro p f Confirm OD HL concentration on spectrophotometer 9 spin Buffer exchange amino oligo Hn Ta 2 STAGE 1 Sulfo S 4FB p y Modify amino oligo w Sulfo S 4FB linker paj i Buffer exchange amp concentrate 4FB modified oligo arp MAN Confirm OD e5 UuL on spectrophotometer and determine 4FB MSR Figure 3 Stage One 1 of the All in One conjugation process illustrates the modification of an amino oligonucleotide using Sulfo S 4FB linker solulink www Solulink com 7 Antibody Oligonucleotide All in One M Conjugation Kit Pe 9g il Confirm IgG concentration on spectrophotometer Resuspend IgG 100 ug at 1 0 mg mL le Ll modification buffer STAGE 2 Buffer exchange into ae ag il S HyNi x HyNic y HyNic modify Fa F dkt HyNic oa Buffer exchange into _ conjugation buffer nae a i i HyNic amp HyNic ki Figure 4 Stage Two 2 of the All in One process illustrates the modification of IgG using S HyNic linker solulink www Solulink com 8 Antibody Oligonucleotid
29. for 10 seconds before the beads have a chance to resettle 8 The conjugate is now bound to the affinity matrix With a P 200 pipette carefully remove and discard the supernatant without disturbing the magnetized bead pellet 9 Immediately add 500 ul Solution D to the bead pellet remove the tube from the magnet and pipette the slurry up and down with a P 1000 several times to wash Never vortex the beads Before the beads resettle place them back on the magnet for 10 seconds Remove and discard the supernatant without disturbing the pellet 10 Repeat step 9 three 3 additional times discarding the wash Supernatant between washes 11 Remove the tube from the magnet and add 100 uL Solution E directly to the bead pellet 12 Using a P 1000 pipette the slurry up and down until the bead pellet adhered to the wall is rinsed to the bottom of the vial Never vortex the beads Set P 1000 to 90 uL when mixing slurry 13 Incubate the settled slurry for 5 minutes away from the magnet 14 Mix the slurry up and down and incubate for another 5 minutes away from the magnet 15 Repeat step 14 one 1 additional time Total conjugate elution time for these three elution incubation periods is 15 minutes 16 Pipette the settled slurry up and down with the P 1000 one last time and immediately place the slurry on the magnet for 10 seconds before the beads have a chance to resettle 17 Without disturbing the pellet carefully transfer the c
30. gonucleotide All in One Conjugation Kit blank until a suitable baseline is obtained Clean the pedestal dry with a Kimwipe 8 Scan a 2 uL drop 4FB Oligo sample on the pedestal by clicking the Measure icon Both black 1 mm and red trace 0 1 mm scans should appear 9 Read the displayed absorbance at A360 black trace by toggling the A2 toggle switch with the mouse until it reaches 360 nm Record the A360 black trace 1 mm path length as displayed in the A2 window 10 Obtain the A260 value red trace 0 1 mm path length by toggling the Max Absorbance toggle switch downward until the A260 from the red trace is just under full scale in the scan window Then using the mouse click the cursor inside the Max Absorbance window and enter a new slightly higher value until the red trace just reaches full scale in the scan window When the red trace is adjusted to full scale read the A260 value displayed in the Max Absorbance window Record the A260 Note numerical entries in the Max Absorbance window can be made in increments of 0 01A units until the red trace exactly reaches full scale 11 Enter the resulting A360 and A260 values into the Conjugation Calculator Section E green cells The calculator then displays the 4FB molar substitution ratio or MSR Section E yellow cell The calculator also displays a warning if the 4FB MSR is too low or too high e g less than 0 5 and greater than 1 1
31. igo to be removed Complete conversion of antibody to conjugate simplifies conjugate purification as illustrated in Figure 2 Antibody oligonucleotide conjugate is purified to near homogeneity by selectively binding the conjugate to a magnetic affinity matrix allowing excess 4FB oligonucleotide to be washed away Affinity bound conjugate is then gently eluted from the matrix and buffer exchanged into long term storage buffer Antibody oligonucleotide conjugates produced with the All in One are stable for up to 1 year when kept at 4 C in storage buffer Pe Lagi HyNic modified IgG HyNic 1 Ka 1 ae a Meg Ld Ea Fy excess 4FB oligo 2 AR conjugate orn excess 4FB oligo magnetic affinity 3 Capture conjugate beads Elute i a h i INS Antibody oligonucleotide conjugate Figure 2 All in One conjugate purification strategy solulink www Solulink com 5 Antibody Oligonucleotide All in One M Conjugation Kit C All in One Conjugation Process The three stages of the conjugation process as summarized below Additional details are illustrated in Figures 3 4 and 5 1 Resuspend and verify oligo concentration spectrophotometer 2 Bufferexchange oligo on spin column 3 Modify amino oligo with Sulfo S 4FB and spin filterconcentrate 4 Verify oligo concentration and determine 4FB MSR spectrophotometer 1 Prepare antibody and verify concentration spectrophotometer 2 Buffer
32. ires the total values provided on the Certificate of Analysis to determine the number of nanomoles OD260 Important If the original Certificate of Analysis is not available for whatever reason the required information can still be generated by pasting and analyzing the known oligo sequence including modifications on Integrated DNA Technologies website using their commercial Oligo Analyzer see link below In these cases since the total OD260 units and nanomoles provided by the vendor on their original Certificate of Analysis is no longer available you must enter the number of OD260 units actually being modified as well as the number of nanomoles represented by that OD260 units into the Conjugation Calculator http www ididna com analyzer Applications OligoAnalyzer Default aspx Important Failure to enter all the required information into the conjugation calculator as stated on the vendor s Certificate of Analysis will disrupt and void subsequent calculator functions Always save the calculator soreadsheet after data entry B Resuspend Amino Oligo 1 Resuspend the amino oligo provided e g minimum of 10 OD260 units into 100 uL Solution A Pipette the solution up and down 30 times using a P 200 pipette barrier tip to completely resuspend the oligo pellet Also rinse the wall of the container with pipette action to insure that any and all oligo pellet material has been resuspended completely 2 1If more than 40 OD260 units are re
33. larified Supernatant 100 ul containing the eluted conjugate to a new labeled 1 5 mL tube solulink www Solulink com 30 Antibody Oligonucleotide All in One Conjugation Kit 18 Repeat step 11 17 one 1 additional time pooling the two 100 uL conjugate fractions together in the same tube 200 uL final volume Buffer exchange the eluted conjugate into storage buffer C Buffer Exchange into Storage Buffer Prepare two spin columns blue cap by twisting off the bottom closure and loosening the cap do not remove the cap Place each spin column into a collection tube provided and mark the top of the blue caps with an indelible pen to identify the conjugate Also place a vertical mark on the side of the spin column as shown on below Label lid w conjugate ID Place pen mark on Side of spin column lt Collection tube 1 Place the two spin columns in the centrifuge and orient the vertical mark on the spin column aiming outward and away from the center of the rotor 2 Centrifuge at 1 500 x g for 1 minute Discard the flow through from each collection tube Each column matrix will appear white in color Place the columns back into new empty collection tubes provided 3 Open each blue cap load 100 uL conjugate to the top of each dry resin bed loosely cap and place them back into their empty collection tube 4 Orient the spin column mark outward as before and centrifuge at 1 500 x g for 2 minutes 5 Elute
34. lid or liquid follow the instructions that apply to your particular sample A Antibody Preparation If the IgG is in a solid lyophilized form 100 ug 1 Add 100 uL Solution B to lyophilized antibody 100 ug solid Cap the sample vial and vortex for 1 minute 2 Open the lid and using a P 100 gently pipette the solution up and down while rinsing the wall of the container from top to bottom Lyophilized antibody can often adhere to the upper walls of a product vial Visually inspect the vial and lid for any residual lyophilized antibody residue that may have become trapped during the vendor packaging process in order to maximize sample recovery Important although careful resuspension of the antibody is tedious notwithstanding it remains a critical step in the conjugation process Antibody vendors rarely overfill product vials so to achieve efficient solulink www Solulink com 24 Antibody Oligonucleotide All in One Conjugation Kit recovery of expensive antibodies great care and diligence is recommended 3 Briefly centrifuge the resuspended antibody at 1 000 x g for 10 seconds io collect the entire liquid contents at the bottom of the vial and proceed to confirm antibody concentration Note if the original IgG product is packaged in a product vial that is too large to fit inside a standard microcentrifuge Such larger vials e g glass vials can first be placed inside a 50 mL disposable conical tube and briefly spun at 10
35. n Solulink recommends that customers always use HPLC purified amino oligonucleotides in this protocol We recommend requesting a mass spectrum to confirm the final quality when available The mass spectrum confirms percent full length purity as well as molecular weight Unambiguous confirmation of amino group As a general rule we do not recommend using crude oligonucleotide preparations to make a conjugate Use barrier pipette tips and good laboratory practices at all times to avoid potential contamination and or cross talk between different oligonucleotide sequences solulink www Solulink com 10 Antibody Oligonucleotide All in One Conjugation Kit F Kit Components Component TS Serap or ont Spin Column Yellow Cap Spin Column Blue Cap G Materials to be Provided by the User Variable high speed microcentrifuge e g Eppendorf 5415D or eguivalent Magnetic single 1 5 ml tube stand e g Ambion AM10026 UV VIS Scanning Spectrophotometer or ND 1000 NanoDrop UV VIS scanning plate reader Bradford Assay Option al Micro volume quartz cuvette 50 100 uL if a NanoDrop 1 5 mL microfuge tubes Bradford protein assay reagents Bio Rad 500 0006 Bovine IgG concentration standards Pierce 23212 Calibrated pipettes P 2 P 10 P 200 and P 1000 and barrier tips Table Top Centrifuge holds 50 mL conical tubes Optional IS not available H Component Storage Conditions 2 8 C re HyNic Reagent
36. ncentration mg mL and total mass of antibody available to be conjugated into the Conjugation Calculator Section F yellow cells A concentration of 1 0 2 mg mL is required to proceed otherwise obtain additional IgG or adjust the concentration to 1 mg mL Note The calculator uses the average known mass extinction coefficient E1 of IgG to calculate protein concentration E1 14 C Buffer Exchange Antibody 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 2 Mark the top of the red cap using an indelible pen to identify the antibody sample Also place a vertical mark on the side of the spin column as shown below Label lid w oligo ID Te Place pen mark on Side of spin column lt Collection tube 3 Place the entire 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 column 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 solulink www Solulink com 27 Antibody Oligonucleotide All in One Conjugation Kit 5 Open the red cap load the antibody sample 100 uL at 1 mg mL to the top of the dry r
37. oligo concentration is in the required range 0 3 to 0 6 OD260 uL proceed to measure the oligo 4FB Molar Substitution Ratio as described in Section of this protocol Leave the 4FB Oligo solution in the concentrator unit until after Section is complete lf the 4FB oligo concentration displayed is greater than 0 6 OD260 uL dilute the 4FB oligo in the concentrator unit by adding the indicated volume of Solution C uL Conjugation Calculator Section D 3 yellow cell to obtain 0 6 OD260 uL then re enter this adjusted value into the Conjugation Calculator Section D green cell Once the 4FB oligo is adjusted to 0 6 OD260 uL proceed to measure the oligo 4FB Molar Substitution Ratio as described in solulink www Solulink com 20 Antibody Oligonucleotide All in One Conjugation Kit Section of this protocol Leave the 4FB Oligo solution in the concentrator unit until after Section is complete Important If the oligo concentration is less than 0 3 OD260 uL at this juncture re concentrate the 4FB oligo in the concentrator unit with additional centrifugation time at 15 000 x g until a volume of 15 20 UL is reached and re confirm the OD260 uL When the required 4FB oligo concentration is obtained re enter the measured value into the Conjugation Calculator Section D green cell and proceed to Section of this protocol Leave the concentration adjusted 4FB Oligo solution in the concentrator unit until after Section is comple
38. omponent Storage Conditions solulink www Solulink com 3 Antibody Oligonucleotide All in One Conjugation Kit A Product Description Each Antibody Oligonucleotide All in One Conjugation Kit provides all the necessary components to generate one 1 antibody oligonucleotide conjugate in just over 10 hours 4 hr hands on The kit requires the user to supply the antibody polyclonal or monoclonal 100 ug and one HPLC purified amino modified oligonucleotide 10 40 OD260 units Kit instructions are specifically designed for researchers with limited or no conjugation experience A special conjugation calculator located on a flash drive is directly integrated with the protocol and avoids the need to perform numerical calculations throughout the procedure Each kit yields between 20 60 ug of highly purified ready to use antibody oligonucleotide conjugate Yield is dependent on both the specific antibody and oligo size Final conjugate concentrations typically range from 0 1 0 3 mg ml B All in One Conjugation Technology 1 Conjugation Chemistry The Antibody Oligonucleotide All in One Conjugation kit uses proprietary HydraLink chemistry to link an antibody to an oligonucleotide as illustrated in Figure 1 The first stage of the process begins with the modification of a 3 or 5 amino modified oligonucleotide using an excess of a HydraLink linker called Sulfo S 4FB This reactive NHS ester incorporates an aromatic aldehy
39. on B green cell The calculator determines the A260 uL as well as the total OD260 oligo units available for conjugation Section B yellow cells A minimum of 10 OD260 and a maximum of 40 OD260 units are required Important If less than 10 OD260 units are recovered after resuspension obtain additional amino oligo If greater than 40 OD260 are resupended transfer an aliquot equivalent to 40 OD260 units into another tube and bring the final volume to 100 uL with Solution A then proceed with the protocol D Buffer Exchange Amino Oligo 1 Prepare a buffer exchange spin column red cap by twisting off the bottom closure and loosening the red cap do not remove the cap Place the spin column into a collection tube provided 2 Mark the top of the spin column red cap using an indelible pen to identify the oligo sequence Using the same marker pen place a single vertical mark anywhere on the side of the spin column as illustrated below Label lid w oligo ID A Io solulink www Solulink com 15 Antibody Oligonucleotide All in One Conjugation Kit 3 Place the spin column assembly into the centrifuge and balance appropriately with an opposing balance tube Orient the vertical mark on the side of the spin column by aiming it 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 bottom of the collection tube The column matrix will appear white in color
40. plete Important If the oligo concentration is less than 0 3 OD260 uL at this juncture re concentrate the 4FB oligo in the concentrator unit with additional centrifugation time at 15 000 x g until a volume of 15 20 UL is reached and then re confirm OD260 uL When the required 4FB oligo concentration is obtained re enter the measured value into the Conjugation Calculator Section D green cell and proceed to Section of this protocol Leave the 4FB Oligo solution in the concentrator unit until after Section is complete Concentration Using a Conventional UV VIS Spectrophotometer 1 Prepare a 1 1000 dilution of the 4FB modified oligo by transferring 1 uL calibrated P 2 pipette from inside the spin filter concentrator body to a tube containing 999 uL molecular grade H2O Label the tube with the appropriate 4FB oligo ID Using a quartz micro cuvette blank the spectrophotometer at 260 nm with molecular grade H O Discard the blank solution from the cuvette Measure the A260 of 1 1000 oligo dilution 4 Enter the resulting A260 into the Conjugation Calculator Section D green cell The calculator will then display the concentration of the 4FB oligo in units of A260 uL Conjugation Calculator Section D 1 yellow cell If the calculator displays YES Section D 2 yellow cell then proceed to step 5 below If the calculator displays FALSE Section D 2 yellow cell proceed to step 6 below When the measured 4FB
41. rcial plate reader solulink www Solulink com 33 Antibody Oligonucleotide All in One M Conjugation Kit B Antibody Oligonucleotide Conjugates Some Examples Mouse mAb oligonucleotide conjugates 44 mer and 22 mer Silverstain 10 SDS PAGE SDS MOPS Protein Molecular Weight Marker 4F B Oligonucleotide 44 mer Crude mouse anti FITC mAb 44 mer conjugation reaction 800 ng Affinity punfied mouse anti FITC mAb 44 mer reaction 800 ng Duplicate of lane 4 Mouse anti FITC mAb 200 ng Crude mouse anti FIT C mAb 22 mer conjugation reaction 800 ng Affinity purified mouse anti FITC mAb 22 mer reaction 800 ng Duplicate of lane 8 1 rA 3 4 5 6 2 8 9 Hamster mAb oligonucleotide conjugate 60 mer solulink www Solulink com 34 Antibody Oligonucleotide All in One Conjugation Kit 250 150 100 15 50 37 5 25 20 AN gt Silver Stain Gel MOPS SDS Buffer 12 NU PAGE SDS Gel Protein molecular weight marker 60 mer 4FB oligonucleotide standard Hamster mAb anti CD3 145 C211 standard 250 ng All in One crude conjugation reaction 60 mer anti CD3 mAb 900 ng protein Affinity purified anti CD3 60 mer antibody oligonucleotide conjugate 900 ng protein C Troubleshooting Guide Possible Cause Recommended Action Poor conjugate yield Poor or undetectable conjugate yield Poor HyNic modification Poor HyNic modification www Solulink com eAmino oligonucleotide may no
42. suspended transfer a volume equivalent to 40 OD260 units into another tube and adjust the final volume to 100 uL with Solution A Store the remaining unused portion of the resuspended amino oligonucleotide at 20 C 3 Vortex the oligo solution for 60 seconds then centrifuge the amino oligonucleotide for 10 seconds at 1 000 x g to collect the full liquid contents at the bottom of the vial Proceed to measure the oligo concentration solulink www Solulink com 13 Antibody Oligonucleotide All in One Conjugation Kit C Measure Amino Oligo Concentration on a Spectrophotometer The amino oligo concentration can measured either on a conventional or micro volume UV VIS scanning spectrophotometer e g NanoDrop ND 1000 When using a conventional spectrophotometer a quartz micro cuvette 50 100 uL is required Follow the corresponding instructions for each type of spectrophotometer as summarized below Micro Volume Spectrophotometer e g NanoDrop ND 1000 1 Determine the concentration OD260 uL of the resuspended amino oligo on a NanoDrop as follows remember to use barrier tips a Prepare a 1 250 dilution of the dissolved amino oligo by transferring 1 uL with a calibrated P 2 pipette into 249 uL molecular grade H O b Select the Nucleic Acid menu option on the NanoDrop and initialize the instrument using molecular grade water c Clean the sample pedestal and blank the instrument with 2uL molecular grade H
43. t be sufficiently 4FB modified Quality and or purity of starting antibody is poor Presence of protein carriers such as BSA or gelatin may be contaminating antibody sample Concentration of S HyNic modification reagent 35 Verify 4FB MSR to insure proper conjugation Concentrate 4FB oligo into the required range 0 3 0 6 OD260 uL elf antibody quality or source are undetermined perform Suitable test such as SDS gel page analysis and or a Bradford protein assay to confirm the purity and quantity of the starting material Remove and purify the antibody sample of all protein carriers such as BSA or gelatin using affinity chromatography or other method before proceeding Make sure to thoroughly dissolve S HyNic reagent before adding it to the solulink Poor HyNic modification Low conjugate and or antibody recovery www Solulink com Antibody Oligonucleotide All in One Conjugation Kit Possible Cause Recommended Action Presence of non protein amine contaminants Improper storage of S HyNic reagent can lead to hydrolysis of the NHS ester initial antibody concentration is low Low buffer exchange spin column recovery volume Low yield during affinity purification of conjugate 36 antibody Use a calibrated pipette to insure accuracy in small volume additions Remove all non protein amine contaminants such as glycine or Tris before modif
44. te I Measure and Quantify 4FB Molar Substitution Ratio The following 4FB Molar Substitution Assay quantifies the amount of 4FB attached to the oligonucleotide The assay is performed by reaction of an aliquot 2 uL of the 4FB oligo solution 0 3 to 0 6 OD260 uL with 2 HP reagent at 37 C for 30 minutes after which the A260 and A360 of the sample is measured on a spectrophotometer This assay insures that the oligo is both 4FB modified and properly buffer exchanged removal of excess Sulfo S 4FB Use the appropriate instructions below depending on the specific type of spectrophotometer available to you e g NanoDrop or Conventional 4FB Molar Substitution Assay NanoDrop 1 Prepare a 2 HP blank solution by adding 2 uL Solution C to 18 uL 2 HP Reagent label 2 HP Blank 2 Prepare a 4FB oligo sample by adding 2 uL 4FB modified oligo 0 3 0 6 OD260 uL to 18 uL 2 HP reagent label 4FB Oligo 3 Incubate 2 HP blank and 4FB Oligo reactions at 37 C for 30 minutes 4 Launch the NanoDrop software and select the UV VIS menu option 5 Initialize the instrument with 2 uL molecular grade water 6 When the scanning window appears make sure the HiAbs feature is clicked on with a check mark in the appropriate box 7 Blank the NanoDrop with 2 uL 2 HP blank solution Reblank to validate a flat baseline If necessary clean the pedestal and re solulink www Solulink com 21 Antibody Oli
45. tion E Dissolve Sulfo S 4FB Reagent 1 Add 25 uL DMF to the vial of Sulfo S 4FB reagent vortex for 30 seconds to resuspend Pipette the DMF solution up and down if necessary to fully resuspend the material adhered to the wall of the vial F Modify Amino Oligo with Sulfo S 4FB Reagent 1 Enter the volume of amino oligo to be modified with Sulfo S 4FB into the Conjugation Calculator Section C green cell solulink www Solulink com 16 Antibody Oligonucleotide All in One Conjugation Kit 2 Add the indicated volume uL of dissolved Sulfo S 4FB reagent as displayed in the Conjugation Calculator Section C yellow cell to the amino oligo vortex to mix Centrifuge at 1000 x g for 10 seconds to collect the entire liquid contents at the bottom of the tube 3 Incubate at room temperature for 2 hours to modify the oligo G Buffer Exchange and Concentrate 4FB Oligo Five minutes prior to the end of the 4FB oligo modification reaction pre wet an Oligo Spin Filter as described in this section Pre Wet Spin Filter 1 Open the lid of an assembled filter unit pre wet the filter membrane inside the concentrator body see image below by adding 500 uL Solution C to the filter membrane Concentrator body Collection tube 2 Pipette the solution up and down using a P 1000 pipette several times without touching or damaging the filter membrane 3 Open the lid to the filter unit and with gloved hands remove
46. ying the antibody with S HyNic reagent Keep and store the S HyNic reagent sealed in the pouch provided below 4 C eConfirm initial antibody concentration prior to S HyNic modification on the spectrophotometer If in doubt perform a Bradford e Dissolve the antibody sample carefully in the original product vial Use a properly calibrated variable speed centrifuge and follow recommended spin speed time Altered spin speeds will adversely compromise recovery e Make sure to follow all the incubation times for binding and elution of conjugate solulink
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