TITLE: Cell DIVE™ Platform | Antibody Purification Chemistry
AUTHORS: Anup Sood, Eric Williams, Liz McDonough

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of the protocol is to purify antibodies that will be conjugated to Cy dyes as per the Cell DIVE™ technology. Affinity chromatography will be used to remove impurities from the vendor antibody to enable conjugation.</div></div>

[STEPS]
?. [Evaluate Need for Purification as per Formulation]
To determine the formulation of the vendor antibody solution refer to the vendor data sheet for product specifications. Refer to Table 7.1, for additives that require the antibody to be purified.  ABCD1FormulationAdditiveBufferNeeds purification2Tissue culture supernatantN/APBSYes3Ascites FluidN/APBSYes4PurifiedStabilizer e.g. BSAPBSYes5PurifiedContains both BSA and GlycerolPBSDilute glycerol content to 10% (5% preferred) before purification6PurifiedStabilizer e.g. TrehalosePBSNo7PurifiedStabilizer e.g. GelatinPBSYes8PurifiedAny amine e.g. glycine etc.PBSIf amine is low molecular weight -Buffer exchange using Amicon filters9PurifiedN/ATRISBuffer exchange using Amicon filters10PurifiedGlycerolPBSBuffer exchange using Amicon filters11PurifiedAzide (up to 0.1%)PBSNo12PurifiedN/APBSIf concentration below 0.5 mg/mL, concentrate using Amicon filters
ABCD1FormulationAdditiveBufferNeeds purification2Tissue culture supernatantN/APBSYes3Ascites FluidN/APBSYes4PurifiedStabilizer e.g. BSAPBSYes5PurifiedContains both BSA and GlycerolPBSDilute glycerol content to 10% (5% preferred) before purification6PurifiedStabilizer e.g. TrehalosePBSNo7PurifiedStabilizer e.g. GelatinPBSYes8PurifiedAny amine e.g. glycine etc.PBSIf amine is low molecular weight -Buffer exchange using Amicon filters9PurifiedN/ATRISBuffer exchange using Amicon filters10PurifiedGlycerolPBSBuffer exchange using Amicon filters11PurifiedAzide (up to 0.1%)PBSNo12PurifiedN/APBSIf concentration below 0.5 mg/mL, concentrate using Amicon filters
?. [Column Equilibration]
Remove the Protein A or Protein G column from  and allow it to equilibrate to room temperature (~1/2hr).
4 °C
Protein A is used to purify rabbit antibodies. Protein G is used to purify all mouse IgG. For purification of antibodies from other species, please consult the product insert.
?. [Column Equilibration]
Screw on the red luer lock adapter in to the top of the column and break off the bottom tab.
?. [Column Equilibration]
Wash the column with  at a flow of 1-2 drops/second.Using a syringe, draw . Attach syringe to the top of the column. Gently push down on the syringe to deliver at flow rate of 1-2 drops/second. Collect flow through in a waste beaker.         o Discard any flow through liquid
[dH2O]
[dH2O]
?. [Column Equilibration]
In order to equilibrate the column, wash with  at a flow of 1-2 drops/second. Using a syringe draw up . Attach syringe to the top of the column. Gently push down on the syringe to deliver 1-2 drops/second flow rate. Collect flow through in a waste beaker.         o Discard any flow through liquid
[1X Binding Buffer]
[1X Binding Buffer]
?. [Antibody Application to the HiTrap Column]
Remove the unpurified antibody from the .
4 °C
?. [Antibody Application to the HiTrap Column]
Retain  for troubleshooting purposes.
[unpurified antibody]
?. [Antibody Application to the HiTrap Column]
Use a 5mL syringe to remove the remaining volume of un-purified antibody from the antibody vial. Use a small gauge needle (23g, usually) to reach the bottom of the container if needed. Retain antibody solution in the syringe.
?. [Antibody Application to the HiTrap Column]
To remove the residual antibody, rinse the antibody vial with .This step is only done if you are not returning the retained 5µg of unpurified antibody (step 7) to the original vial.If you are returning the retained 5µg of unpurified antibody (step 7) to the original vial, use a separate Eppendorf vial to draw up the 1X binding buffer from - so as to not contaminate the stock vial.
[1X Binding Buffer]
?. [Antibody Application to the HiTrap Column]
Using the same 5 mL syringe, remove the wash volume from the antibody vial.
?. [Antibody Application to the HiTrap Column]
Remove the needle, invert the syringe and remove any air bubbles by gently flicking the sides of the syringe.
?. [Antibody Application to the HiTrap Column]
Apply the antibody sample to the column at a rate of 1-2 drop/second, collecting flow through liquid in a 15 mL conical tube. Minimize the amount of air pushed through the columnSave the flow through for potential troubleshooting. Discard after 24 hours - typically discarded after adequate recovery of the antibody is shown.
?. [Wash Unbound proteins from the HiTrap column]
Prepare five 1.5 mL clear eppendorf tubes by labeling them W1-W5 (Wash 1-Wash 5).
?. [Wash Unbound proteins from the HiTrap column]
Measure  in a new 5 mL syringe.
[fresh 1X Binding Buffer]
?. [Wash Unbound proteins from the HiTrap column]
Apply the 1X Binding Buffer to the column at a flow rate of 1-2 drop/second.
?. [Wash Unbound proteins from the HiTrap column]
Collect  in the labeled tubes starting with W1 and proceed in sequential order.
[fractions of the wash]
?. [Wash Unbound proteins from the HiTrap column]
Vortex each wash for . Briefly centrifuge to remove any liquid from the underside of the cap and upper parts of the tube.
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
Open the Nanodrop program from the computer connected to the Nanodrop.
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
From the main screen select “Proteins A280” program.
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
Within the “Protein A280” program, select Type BSA, refer to Figure 7.5.1
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
To blank the system, add  to the pedestal.
[fresh 1X Binding Buffer]
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
Select “Blank.”
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
After blanking is completed, wipe pedestal with a Kimwipe or other low lint wipe.
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
To ensure a stable baseline add  to the pedestal.
[fresh 1X Binding Buffer]
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
Select “Measure” Refer to Figure 7.5.2A stable baseline (right) shows a level line at zero with little signal noise. An unstable unacceptable baseline appears tilted.
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
After measuring, wipe pedestal with a Kimwipe.
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
Once a stable baseline is achieved measure the absorbance of each wash step in triplicate
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
After each measurement, wipe pedestal with a kimwipe.
?. [Use the NanoDrop to confirm the complete removal of unbound protein]
The Nanodrop will calculate the protein concentration of each reading. If a protein concentration at >0.03 mg/mL is present in Wash 5, continue collecting  until the concentration in the last wash reads
[wash fractions]
?. [Preparing Amicon 30K filtration device]
Remove Amicon 30K filter from its packaging.
?. [Preparing Amicon 30K filtration device]
Fill the filter to the top with dH2O and cap.
?. [Preparing Amicon 30K filtration device]
Gently invert the filter 5 times.
?. [Preparing Amicon 30K filtration device]
Spin the filter at  in the centrifuge.
Centrifuge: 3000 33, 10 min
?. [Preparing Amicon 30K filtration device]
After  discard the flow through liquid.
?. [Preparing Amicon 30K filtration device]
Repeat steps 31-34 one more time.
?. [Eluting the purified antibody from the HiTrap Column]
Prepare five 1.5 mL eppendorf tubes by labeling them E1-E5 (Elution1-Elution5).
?. [Eluting the purified antibody from the HiTrap Column]
Measure  and pipette into tubes E2-E5.
[fresh 1X Neutralizing buffer]
?. [Eluting the purified antibody from the HiTrap Column]
Draw  into a 5 mL syringe.
[fresh 1X Elution Buffer]
?. [Eluting the purified antibody from the HiTrap Column]
Apply the  to the HiTrap Column at a flow rate of 1-2 drops/second.
[1X Elution Buffer]
?. [Eluting the purified antibody from the HiTrap Column]
Collect  in the labeled tubes starting with E1 and proceed in sequential order.
[fractions of the eluted antibody]
?. [Eluting the purified antibody from the HiTrap Column]
Vortex each fraction for .Alternately, invert tube and flick so as to mix.Centrifuge briefly so as to remove solution from the underside of the cap and the upper parts of the tube.
?. [Confirming the complete removal of purified antibody via NanoDrop]
From the opened Nanodrop program, select “Proteins A280” program.
?. [Confirming the complete removal of purified antibody via NanoDrop]
Within the “Protein A280” program, select protein Type: IgG. Refer to Figure 7.8.1.
?. [Confirming the complete removal of purified antibody via NanoDrop]
To blank the system add  to the pedestal.
[Neutralized Elution Buffer]
?. [Confirming the complete removal of purified antibody via NanoDrop]
Select “Blank.”
?. [Confirming the complete removal of purified antibody via NanoDrop]
After blanking has completed, wipe pedestal with a Kimwipe.
?. [Confirming the complete removal of purified antibody via NanoDrop]
To ensure a stable baseline add  to the pedestal.
[Neutralized Elution Buffer]
?. [Confirming the complete removal of purified antibody via NanoDrop]
Select “Measure” After measuring, wipe pedestal with a Kimwipe. If a satisfactory baseline isn't achieved, return to step 41.Refer to Figure 7.5.2.
?. [Confirming the complete removal of purified antibody via NanoDrop]
In subsequent steps, recover the antibody after each measurement by drawing it back in to the pipette and placing it back into the assigned eppendorf tube.
?. [Confirming the complete removal of purified antibody via NanoDrop]
Measure each fraction in triplicate, generally the first fraction will be free of any protein, elution 2 and 3 will contain pure antibody, and elution 4 and 5 will be free of any protein. If fraction 5 contains a protein concentration > 0.03 mg/mL continue collecting  until no more protein is eluted.
[fractions]
?. [Concentration and Buffer Exchange of the purified antibody]
Combine all elution fractions with a protein concentration of >0.03 mg/mL into the prepared Amicon 30K filter.
?. [Concentration and Buffer Exchange of the purified antibody]
Fill the filter to the manufacturer's recommended level with fresh 1X PBS.
?. [Concentration and Buffer Exchange of the purified antibody]
Apply the cap and invert the filter 5 times to completely mix the PBS and antibody solution.
?. [Concentration and Buffer Exchange of the purified antibody]
Place the solution in the centrifuge and spin at .
Centrifuge: 3000 33, 10 min
?. [Concentration and Buffer Exchange of the purified antibody]
After , check the filter to ensure the volume will lead to a calculated concentration of  or higher. Example: If you purify , the maximum volume to spin down to would be  to get a concentration of . If it has not, continue spinning at  for  intervals until the appropriate volume is achieved.
800 µg
800 µl
Centrifuge: 3000 33
?. [Concentration and Buffer Exchange of the purified antibody]
When the solution is at or above a calculated concentration of , remove the bottom tube and transfer the flow through liquid to a separately labeled 15 mL conical tube.
?. [Concentration and Buffer Exchange of the purified antibody]
Repeat steps 52-55, three more times for a total of 4 spins.
?. [Concentration and Buffer Exchange of the purified antibody]
Following the 4th spin, remove the filter from the 15 mL tube.
?. [Concentration and Buffer Exchange of the purified antibody]
Remove the purified antibody from the filter with a 200uL pipette.
?. [Concentration and Buffer Exchange of the purified antibody]
Transfer to a clean 1.5 mL eppendorf tube labeled with antibody name.
?. [Concentration and Buffer Exchange of the purified antibody]
Wash the filter with  and then repeatedly pipette to wash each side of the filter.
[fresh 1X PBS]
?. [Concentration and Buffer Exchange of the purified antibody]
Remove the wash and combine with purified antibody in 1.5 mL Eppendorf tube from step 60.
?. [Concentration and Buffer Exchange of the purified antibody]
Close the lid of the Eppendorf tube and mix the solution gently but well, centrifuging briefly to remove liquid from the under-side of the cap.
?. [Determining final yield via NanoDrop]
From the opened Nanodrop program, select “Proteins A280” program.
?. [Determining final yield via NanoDrop]
Within the “Protein A280” program, select protein Type: IgG, Selecting protein Type: IgG.
?. [Determining final yield via NanoDrop]
To blank the system, add  to the pedestal.
[fresh 1X PBS]
?. [Determining final yield via NanoDrop]
Select “Blank.”
?. [Determining final yield via NanoDrop]
After blanking, wipe pedestal with a kimwipe.
?. [Determining final yield via NanoDrop]
Select “Measure.” After measuring, wipe pedestal with a kimwipe.If the baseline is poor, return to step 66.Refer to Figure 7.5.2.
?. [Determining final yield via NanoDrop]
Measure the purified antibody 5 times.
?. [Determining final yield via NanoDrop]
Take a volumetric reading of the final purified antibody with a pipette and record volume.
?. [Determining final yield via NanoDrop]
Calculate %Yield ((Ab concentration (µg/µL)*Volume (µL))/Initial Ab amount (µg)). If the initial antibody amount is not known, the %yield cannot be calculated.
?. [Determining final yield via NanoDrop]
Dilute purified antibody with 0.65% Sodium Azide (Volume of purified antibody/12) to a final concentration of 0.05% Sodium Azide.
?. [Determining final yield via NanoDrop]
Label the vial of purified antibody with the biomarker name, vendor name, catalog number, lot number, date purified and final concentration.
?. [Stabilizing the HiTrap Column with Column Storage Solution]
The HiTrap columns can be re-used for up to 1 year; however, only antibodies from the same vendor and with the same clone can be re-applied to a previously used column.
?. [Stabilizing the HiTrap Column with Column Storage Solution]
Measure , typically 20% ethanol in water, with a 5mL syringe.
[Column Storage solution]
?. [Stabilizing the HiTrap Column with Column Storage Solution]
Apply solution to HiTrap column at a rate of 1-2 drops/second and discard flow through liquid.
?. [Stabilizing the HiTrap Column with Column Storage Solution]
Label the column using a tough tag labeled with the antibody name, clone, vendor, catalog number, protein type (A or G), and date opened.
?. [Stabilizing the HiTrap Column with Column Storage Solution]
Re-attach the top and bottom enclosures and store at  for up to 1 year.
4 °C
?. [Troubleshooting]
If antibody yield is
?. [Troubleshooting]
Also, when there is question about stabilizer such as BSA present in the antibody supplied from vendor. Please record UV spectrum of the antibody and make sure it looks like below with A260/A280=~0.70. If BSA is present A280 will go up and the ratio will drop down from 0.70 to any number depending on the amount of BSA present in the sample.
?. [Stabilizing the HiTrap Column with Column Storage Solution]
Measure  with a 5mL syringe.
[water]
?. [Stabilizing the HiTrap Column with Column Storage Solution]
Apply water to HiTrap column at a rate of 1-2 drops/second and discard flow through liquid.