Recombinant Antibody Affinity Purification with Protein A or Protein G
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Disclaimer
We have listed the specific equipment, reagents, and methods that we use in our lab at Addgene. Equipment and reagents from other vendors should produce similar outcomes when using these protocols. However, please be aware that your protocol may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or recommend specific products or equipment. Inclusion of this information is solely for transparency intended to support reproducibility in science.
Abstract
This protocol describes how to affinity purify recombinant antibodies from cell culture supernatant using Protein A or Protein G columns.
Before start
See the Materials section for preparation of necessary stock solutions.
Warm the affinity column, binding buffer and elution buffer to room temperature before use.
Wipe down all pipettes and equipment with 10% bleach prior to use.
Steps
Affinity chromatography
Equilibrate filtered tissue culture supernatant containing the recombinant antibody to Room temperature (see Transfection for Recombinant Antibody protocol).
Add 1 part Protein A/G binding buffer to 1 part tissue culture supernatant and mix well.
Uncap the Protein G Gravitrap or rProtein A Gravitrap columns and gently pour off the ethanol storage buffer.
Tightly attach LabMate PD10 Buffer Reservoirs to the top of the Gravitrap column.
Attach the Gravitrap column to a clamp on a clamp stand.
Use scissors to cut the bottom of the Gravitrap column at the indentation in the tip.
Equilibrate the column with 10mL of Room temperature Protein A/G binding buffer.
Collect the flow-through in a 50 mL conical tube and dispose after use.
Carefully pour the filtered and diluted cell culture supernatant into the Gravitrap column.
Collect the flow through in a 250 mL bottle.
Repeat steps 9-10 such that the supernatant passes through the column a total of 2 times.
Wash the column with 25mL of Protein A/G binding buffer 2x.
Add 50µL of 1 M Trizma hydrochloride 9 to 10 microcentrifuge tubes
Cap the Gravitrap columns at the bottom.
Add 5mL of Pierce IgG Elution Buffer to the capped Gravitrap column and incubate for 0h 10m 0s at Room temperature.
Uncap the column and collect 500µL fractions into the tubes containing 50µL 1 M Trizma hydrochloride 9.
Cap the tubes and vortex for 0h 0m 5s to mix.
Determine the protein concentration of each fraction on the NanoDrop Spectrophotometer using the A280 IgG setting.
Combine all eluates with measurable protein.
Determine the protein concentration of the pooled sample on the NanoDrop Spectrophotometer using the A280 IgG setting.
If the concentration of the pooled sample is above 1.0 mg/mL proceed to Step 25 with a buffer exchange using a Zeba Spin 7 kDa MWCO desalting column.
If the concentration of the pooled sample is below 1.0 mg/mL proceed to Step 42 with a buffer exchange/concentration using an Amicon 30 kDa MWCO buffer exchange column.
(Optional) Regenerate the column by washing with 25mL of Protein A/G binding buffer and store in 20% ethanol at 4°C.
Buffer exchange
Choose Option 1 (Buffer exchange using a desalting column) or Option 2 (Buffer exchange and concentration with an Amicon column) based on the concentration of the pooled sample above.
Option 1 Buffer exchange using a desalting column
The 10 mL Zeba Spin Desalting Columns can accommodate up to 4mL of recombinant antibody sample.
Twist off the 10 mL Zeba Spin Desalting Column’s bottom closure and loosen cap.
Place column in a 50 mL conical collection tube.
Centrifuge column at 1000x g,0h 0m 0s for 0h 2m 0s to remove storage solution.
Place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge with the mark facing outward in all subsequent centrifugation steps.
Discard the flow through into a waste container.
Add 5mL PBS to the column.
Centrifuge at 1000x g,0h 0m 0s for 0h 2m 0s to remove PBS.
Repeat Steps 31-32 3x, discarding buffer from the collection tube each time.
Place column in a new 50 mL LoBind collection tube.
Slowly apply the sample to the center of the compact resin bed.
Allow the resin bed to fully absorb the sample.
Centrifuge at 1000x g,0h 0m 0s for 0h 2m 0s to remove PBS.
Discard column after use.
Determine antibody concentration on the NanoDrop Spectrophotometer.
Dilute antibody to 1 mg/mL with PBS if needed.
For long term storage, add sterile sodium azide to 1 mM.
Option 2 Buffer exchange and concentration with an Amicon column
Apply 15mL of PBS to the reservoir of a Amicon Ultra-4 30 kDa MWCO column.
Incubate for 0h 10m 0s at Room temperature.
Centrifuge at 3100x g,0h 0m 0s for 0h 8m 0s.
Discard the flow through into a waste container.
Add the recombinant antibody sample to the reservoir of the column.
Fill the remaining space in the column with PBS and pipette several times to mix.
Centrifuge at 3100x g,0h 0m 0s for 0h 8m 0s.
Discard the flow through into a waste container.
Repeat steps 47-49 at least 4 additional times to ensure a full buffer exchange.
Discard the flow through into a waste container.
Fill the remaining space in the column with PBS and pipette several times to mix.
Centrifuge at 3100x g,0h 0m 0s for 0h 8m 0s.
Remove a small aliquot of your sample from the filter reservoir to check the concentration on the NanoDrop Spectrophotometer to see if the sample has reached the desired concentration.
If the sample concentration is still too low, repeat the centrifugation until the volume of the sample is reduced enough to reach the desired concentration.
Gently transfer the sample from the reservoir to a LoBind tube.
Determine the concentration of the sample on the NanoDrop Spectrophotometer.
For long term storage, add sterile sodium azide to 1 mM.
