Affinity purification of antibodies
- Torsten Waldminghaus 09:31, 3 November 2008 (EST):
This protocol describes purification of a polyclonal antibody that might be needed for immuno fluorescens microscopy or co-immuno precipitations.
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Coupling to activated beads
The first step is to couple the antiben to activated beads. In the following procedure CNBr-activated beads from Sigma are used and the procedure is taken from there Product information (sigma-aldrich)
- Dissolve protein to be coupled in 0.1 M NaHCO3 buffer containing 0.5 M NaCl, pH 8.3-8.5 (about 5-10 mg protein per mL of gel). Note: Other buffers can be used, but avoid amine-containing buffers such as Trizma or other nucleophiles (buffers with amino groups) which will react with the binding sites.
- Wash and swell cyanogen-bromide activated resin in cold 1 mM HCl for at least 30 minutes. A total of 200 ml per gram of dry gel is added in several aliquots. Remove the supernatant (contains lactose) by gentle suction in a Büchner or suction funnel between successive additions. Note: Lactose is necessary to stabilize the beads during freeze-drying, but it will interfere with binding if present during coupling. The use of HCl preserves the activity of the reactive groups which hydrolyze at high pH.
- Wash the resin with distilled water, 5 - 10 column volumes, then wash the resin with the NaHCO3/NaCl coupling buffer (5 ml per gram dry gel) and immediately transfer to a solution of the ligand in coupling buffer. Note: The reactive groups hydrolyze in basic solution!
- Mix protein with gel for 2 hours at room temperature or overnight at 4°C. Use a paddle stirrer or end-over-end mixer, but not a magnetic stir bar (which may grind beads).
- Wash away unreacted ligand using NaHCO3/NaCl coupling buffer described above.
- Block unreacted groups with either 1 M ethanolamine or 0.2 M glycine, pH 8.0 for 2 hours at room temperature or 16 hours at 4°C.
- Wash extensively to remove the blocking solution, first with basic coupling buffer at pH ≈ 8.5, then with acetate buffer (0.1 M, pH 4) containing NaCl (0.5 M).
- Complete this wash cycle of high and low pH buffer solutions four or five times.
- If the resin is to be used immediately, equilibrate it in buffer. If not, store the resin in 1.0 M NaCl at 2-8°C with a suitable bacteriostat.
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