Immunoaffinity Chromatography Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Immunoaffinity Chromatography Protocol

Solutions:

Wash buffer
20mM Tris
0.2% Triton
or
1M Tris 1mL
20% Triton 500uL
water up to 50mL

Elution buffer
same solution as wash buffer, but with 4% SDS (perhaps also protease inhibitors if required, but not likely necessary, since the other proteins have been purified out)

1. Spin down cell debris in a proper ultracentrifuge at 100,000 x g for 10 min.
2. If have enough sample, take out enough sample to be able to run the pre-column fraction three times on a gel (e.g.100uL)
3. incubate the sample in a sufficient quantity of beads that have been covalently conjugated to the antibody
4. if complete binding to the beads/antibody is required, incubate overnight
5. spin down the beads for a few seconds in a centrifuge, and take out some sample to run the unbound fraction on a gel
6. resuspend the beads in the remaining solution and place into a spin column (eg from Millipore that have filters of the correct pore size that prevent the beads from being spun to the bottom of the tube)
7. spin down for a few seconds the supernatant and dump out the liquid that comes out the bottom of the filtrate
8. place the column on ice
9. add in wash buffer to the column (or, it may be necessary to wash out the tube that the sample came from with wash buffer and use that to place on the column)
10. wash out the column for at least 5-6 more times by repeating the spinning and adding new wash buffer (some people save the wash buffer to analyze if any protein has eluted during that step)
11. before adding the elution buffer, spin down and remove the wash buffer
12. if using SDS in the elution buffer, shake at room temperature (~250rpm) for ~15 minutes
13. spin down, keep the eluted buffer (which contains your sample), add more elution buffer to shake another 5 minutes
14. spin down again; additional shakings may be carried out if necessary