Protein A and Protein G Purification of Antibodies
Abstract
Protein A and Protein G are immunoglobulin-binding proteins expressed in Staphylococcus aureus and Streptococcus sp., respectively, that have been adapted for use in purifying large amounts of IgG. They are available covalently attached to affinity resins such as 4% cross-linked agarose, making them suitable for low-pressure antibody isolation. Protein A is not recommended for the isolation of mouse mAbs because it lacks affinity for mouse IgG1, or for the isolation of antibodies from sheep, goat, chicken, hamster, or rat. IgGs from most species bind to Protein G at near physiological pH and ionic strength with a higher affinity than IgG binding to Protein A. Therefore, the pH required to dissociate bound IgG is lower, resulting in the loss of activity for some antibodies. If this is observed, Protein A may be an alternative if the IgG from the species being isolated can be purified using Protein A. Neither Protein A nor Protein G can be used for the isolation of chicken antibodies.
MATERIALS
Reagents
Antibody-containing solution
Glycine (0.1 m, pH 2 or 3)
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A lower pH is used to elute IgG from Protein G than from Protein A. To elute IgG from Protein A, use 0.1 m glycine (pH 3). To elute IgG from Protein G, use 0.1 m glycine (pH 2).
Protein A or Protein G agarose or equivalent
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Both Protein A and Protein G covalently attached to cross-linked agarose or other matrices are available from many different vendors. The typical binding capacity of Protein A is anywhere from 15 to 35 mg of human IgG per milliliter of resin. The typical binding capacity of Protein G is 10–25 mg of human IgG or 5–10 mg of mouse IgG per milliliter of resin.
Sodium azide
Tris-buffered saline (TBS, 0.1 m) or Phosphate-buffered saline (PBS) (pH 7.4)
Tris-HCl (2 m, pH 8)
Equipment
Centrifuge to clarify the serum
Dialysis tubing
pH meter
Small column or disposable syringe fitted with a coarse frit
UV spectrophotometer
METHOD
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1. Wash the Protein A or Protein G resin with at least 10 column volumes of 0.1 m TBS or 1× PBS.
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2. Dilute the serum 1:1 with the buffer used to wash the column. Centrifuge the diluted serum at 10,000g for 15 min before loading onto the column to remove cellular debris.
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3. Pass the serum over the column at least two times using either gravity or a pump.
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4. Wash the column extensively until no protein can be detected in the eluent. Save the flowthrough until the final product quality and yield have been determined.
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5. For Protein A, elute bound antibody using 0.1 m glycine (pH 3), and collect the fractions in tubes containing 2 m Tris-HCl (pH 8) (so that the Tris is 15% of the final volume per fraction) to neutralize the eluted antibody. For protein G, elute bound antibody using 0.1 m glycine (pH 2), and collect the fractions in tubes containing 2 m Tris-HCl (pH 8) (so that the Tris is 25% of the final volume per fraction) to neutralize the eluted antibody.
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Because a lower pH is used to elute IgG from Protein G than from Protein A, care must be taken to ensure rapid fraction collection and neutralization.
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See Troubleshooting.
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6. Dialyze the antibody against two or three changes of TBS or PBS over 24–48 h (≥100× the volume of the antibody solution).
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7. Regenerate the column with 10–20 column volumes of 0.1 m TBS or 1× PBS. If the column is to be stored, add 0.02% sodium azide and store the column at 4°C.
TROUBLESHOOTING
Problem (Step 5): The antibody fails to elute from the column.
Solutions:
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Regenerate and wash the column exhaustively before reapplying the serum.
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If the resin has been used several times, proteolysis or leaching may have reduced the amount of Protein A/G bound to the column. Improper storage could result in bacterial contamination and degradation of the Protein A/G. In either case, pour a new column.
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Protein A/G does not bind all antibody types and subclasses. Ensure that the type of antibody to be purified will bind to Protein A/G.
