Peptide Affinity Purification of Antibodies
Abstract
This protocol describes antibody purification using a peptide affinity column. Peptides can be designed that use naturally occurring cysteines within the protein target's primary sequence, or a cysteine can be added to either end of the peptide to provide free thiols for attachment. The peptides can then be covalently attached to resins bearing thiol-reactive linkers. The most commonly used thiol-reactive moieties are iodoacetyl and maleimide, both of which react selectively with peptides containing cysteine thiols. Although gravity can be used to cycle the antibody solution (e.g., serum) over the column (it is recommended that the antibody be cycled multiple times to obtain maximal yield), the use of a pump to apply the serum to the column in a continuous flow manner improves the yield of antibody. Similarly, washing the column after application of the antibody without and with 0.5 m NaCl should be performed with at least 20 column volumes.
MATERIALS
Reagents
Glycine (0.1 m, pH 2 and/or pH 3)
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See Step 6.
High-salt wash buffer (Tris-buffered saline [TBS, 0.1 m] or Phosphate-buffered saline [PBS] [pH 7.4] containing 500 mm NaCl)
Peptide bound to agarose or equivalent (from Protocol: Conjugation of Peptides to Thiol-Reactive Gel for Affinity Purification of Antibodies [Fishman and Berg 2019])
Solution (e.g., serum) containing antipeptide antibodies
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It is critical to the overall recovery and purity of the final antibody fraction that before loading onto the column the serum, antibody solution, ascites, or other source of antibody be centrifuged or filtered through a 0.45-μm filter to remove debris and other flocculent material. Serum should be diluted 1:1 with PBS (or TBS if preferred) before filtration.
Tris-buffered saline (TBS, 0.1 m) or Phosphate-buffered saline (PBS) (pH 7.4)
Tris-HCl (1 m, pH 8.8)
Equipment
Dialysis tubing
pH meter
Pump (recommended)
Small column or disposable syringe fitted with a coarse frit
UV spectrophotometer
METHOD
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1. At room temperature, pack the desired amount of resin into a column. Wash the column with 10–20 column volumes of either 0.1 m PBS or TBS (pH 7.4).
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2. Dilute the antibody solution with an equal volume of 0.1 m PBS or TBS (pH 7.4). Remove debris from the solution by centrifugation or filtration through a 0.45-μm filter.
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3. Load the clear solution onto the column. Pass the material over the column four to six times at room temperature, or use a pump and circulate the antibody overnight at 4°C.
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4. Wash the column with 15–20 column volumes of the buffer used in Step 1.
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5. Wash with 20 volumes of high-salt wash buffer. Monitor the presence of protein in the output at A280 nm. When the column output is free from protein, the antibody can be eluted.
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6. Elute the antibody with 100 mm glycine (pH 3). (For more tightly bound antibodies use pH 2.) Collect the fractions in tubes containing 1 m Tris-HCl (pH 8.8) to neutralize the eluted antibody. If the glycine is at pH 3, then the Tris should be 15% of the final volume per fraction. If the glycine is at pH 2, then the Tris should be 25% of the final volume per fraction.
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7. Measure the protein concentration at A280 nm, and pool the fractions containing the eluted antibody.
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See Troubleshooting.
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8. Dialyze the antibodies against two or three changes of 0.1 m PBS over 24–48 h (≥100× the volume of the antibody solution). Adjust the solution to the desired protein concentration.
TROUBLESHOOTING
Problem (Step 7): Antibody does not elute from the column or elutes poorly at low pH. Similarly, antibody elutes at low pH, but no activity is detected.
Solution: Elute antibody at high pH using 100 mm triethylamine (pH 11.5). Neutralize the eluent using a variety of 1 m buffers at pH 6. Other elution reagents to try are 3 m MgCl2 in 20 mm Tris-HCl (pH 7.5) or chaotropic salts such as 2 m KSCN. In the case of KSCN, it is critical to separate the antibody from the chaotrope as quickly as possible using a G-25 column, for example.
Footnotes
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From the Antibodies collection, edited by Edward A. Greenfield.
