TITLE: Recombinant Antibody Affinity Purification with Protein A or Protein G
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
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.

[GUIDELINES]
General Considerations
Protein A and Protein G have varying affinities for species and isotypes. Refer to the manufacturer’s guidelines to determine the best protein for your antibody.

Workflow Timeline
Day 1: Purify antibody
Day 2 or later: Buffer exchange

Tips and Troubleshooting:
We recommend wiping down all pipettes and equipment with 10% bleach prior to use. 

[STEPS]
SECTION: Affinity chromatography
2. 
Add 1 part Protein A/G binding buffer to 1 part tissue culture supernatant and mix well.
SECTION: Affinity chromatography
1. Equilibrate filtered tissue culture supernatant containing the recombinant antibody to Room temperature (see Transfection for Recombinant Antibody protocol).
 


SECTION: Affinity chromatography
3. Uncap the Protein G Gravitrap or rProtein A Gravitrap columns and gently pour off the ethanol storage buffer.
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4. Tightly attach LabMate PD10 Buffer Reservoirs to the top of the Gravitrap column.
 

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5. Attach the Gravitrap column to a clamp on a clamp stand.
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6. Use scissors to cut the bottom of the Gravitrap column at the indentation in the tip.
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7. Equilibrate the column with 10 mL of Room temperature Protein A/G binding buffer.

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8. Collect the flow-through in a 50 mL conical tube and dispose after use.
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9. Carefully pour the filtered and diluted cell culture supernatant into the Gravitrap column.
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10. Collect the flow through in a 250 mL bottle.
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11. Repeat steps 9-10 such that the supernatant passes through the column a total of 2 times.
 

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12. Wash the column with 25 mL of Protein A/G binding buffer 2x.

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13. Add 50 µL of 1 M Trizma hydrochloride pH 9 to 10 microcentrifuge tubes

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14. Cap the Gravitrap columns at the bottom.
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15. Add 5 mL of Pierce IgG Elution Buffer to the capped Gravitrap column and incubate for 10 min at Room temperature.

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16. Uncap the column and collect 500 µL fractions into the tubes containing 50 µL 1 M Trizma hydrochloride pH 9.

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17. Cap the tubes and vortex for 5 s to mix.

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18. Determine the protein concentration of each fraction on the NanoDrop Spectrophotometer using the A280 IgG setting.
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19. Combine all eluates with measurable protein.
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20. Determine the protein concentration of the pooled sample on the NanoDrop Spectrophotometer using the A280 IgG setting.
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21. 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.

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22. 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.

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23. (Optional) Regenerate the column by washing with 25 mL of Protein A/G binding buffer and store in 20% ethanol at 4 °C.



SECTION: Option 1 Buffer exchange using a desalting column
26. Twist off the 10 mL Zeba Spin Desalting Column’s bottom closure and loosen cap.
SECTION: Option 1 Buffer exchange using a desalting column
27. Place column in a 50 mL conical collection tube.
SECTION: Option 1 Buffer exchange using a desalting column
28. Centrifuge column at 1000 x g for 2 min to remove storage solution.

SECTION: Option 1 Buffer exchange using a desalting column
29. 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.
 

SECTION: Option 1 Buffer exchange using a desalting column
30. Discard the flow through into a waste container.
SECTION: Option 1 Buffer exchange using a desalting column
31. Add 5 mL PBS to the column.

SECTION: Option 1 Buffer exchange using a desalting column
32. Centrifuge at 1000 x g for 2 min to remove PBS.

SECTION: Option 1 Buffer exchange using a desalting column
33. Repeat Steps 31-32 3x, discarding buffer from the collection tube each time.
SECTION: Option 1 Buffer exchange using a desalting column
34. Place column in a new 50 mL LoBind collection tube.
SECTION: Option 1 Buffer exchange using a desalting column
35. Slowly apply the sample to the center of the compact resin bed.
 


SECTION: Option 1 Buffer exchange using a desalting column
36. Allow the resin bed to fully absorb the sample.
SECTION: Option 1 Buffer exchange using a desalting column
37. Centrifuge at 1000 x g for 2 min to remove PBS.
SECTION: Option 1 Buffer exchange using a desalting column
38. Discard column after use.
SECTION: Option 1 Buffer exchange using a desalting column
39. Determine antibody concentration on the NanoDrop Spectrophotometer.
SECTION: Option 1 Buffer exchange using a desalting column
40. Dilute antibody to 1 mg/mL with PBS if needed.
SECTION: Option 1 Buffer exchange using a desalting column
41. For long term storage, add sterile sodium azide to 1 mM.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
42. Apply 15 mL of PBS to the reservoir of a Amicon Ultra-4 30 kDa MWCO column.

SECTION: Option 2 Buffer exchange and concentration with an Amicon column
43. Incubate for 10 min at Room temperature.

SECTION: Option 2 Buffer exchange and concentration with an Amicon column
44. Centrifuge at 3100 x g  for 8 min.

SECTION: Option 2 Buffer exchange and concentration with an Amicon column
45. Discard the flow through into a waste container.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
46. Add the recombinant antibody sample to the reservoir of the column.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
47. Fill the remaining space in the column with PBS and pipette several times to mix.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
48. Centrifuge at 3100 x g  for 8 min.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
49. Discard the flow through into a waste container.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
50. Repeat steps 47-49 at least 4 additional times to ensure a full buffer exchange.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
51. Discard the flow through into a waste container.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
52. Fill the remaining space in the column with PBS and pipette several times to mix.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
53. Centrifuge at 3100 x g  for 8 min.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
54. 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.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
55. 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.
 

SECTION: Option 2 Buffer exchange and concentration with an Amicon column
56. Gently transfer the sample from the reservoir to a LoBind tube.
 

SECTION: Option 2 Buffer exchange and concentration with an Amicon column
57. Determine the concentration of the sample on the NanoDrop Spectrophotometer.
SECTION: Option 2 Buffer exchange and concentration with an Amicon column
58. For long term storage, add sterile sodium azide to 1 mM.
SECTION: Option 1 Buffer exchange using a desalting column
25. The 10 mL Zeba Spin Desalting Columns can accommodate up to 4 mL of recombinant antibody sample.
 


SECTION: Buffer exchange
None. 
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24. 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.