I want to use an antibody to pull the antigen out for sequencing. The size of the antigen is about 25 kD. I used protein A agarose pull down, the problem is that the molecular weight of IgG light chain is too close to the targeted protein. So what the else way should I do to get the purified target protein?
If you leave out the reducing agent from the sample buffer when you elute the protein from the beads, the light chain will remain attached to the heavy chain and the whole 150 kDa immunglobulin molecule will be easily separated from your 25 kDa protein on SDS-PAGE.
If you absolutely need reducing buffer to elute perhaps try 2D electrophoresis separation of the Ig light chain and taget protein.
You may also want to first bind your antibody to Protein A beads; then to do a short crosslinking step using eg DMP (Dimethylpimelimidate).
THe x-linking may also interfere with the affinity and specificity of the antibody since it unspecifically reacts with amino-groups all over the place, but once optimized this prevents the antibody of eluting from the beads no matter which agent/condition you use. (This should be even resistant to boiling in reducing SDS-PAGE-sample buffer).
I have done this many years ago. In case you cannot find a protocol in google, I can try to find my old notes
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