Does anyone have experience in using membrane proteins solubilized by detergents to coat an ELISA plate? I am trying this by using CHAPS detergent which I expect to get rid of dialyzing the sample, since the presence of it would prevent the proteins from adsorbing to the plate.
If this works which type of immunoplate should I use to design my ELISA assay?
Any regular plate (polystyrene-based) will do the trick. My concern is the remotion of chaps, which could promote protein aggregation given its hydrophobic nature. I think you should maintain some detergent to prevent this even though it will reduce protein adsorption.
If you set up a sandwich ELISA, the presence of the detergent shouldn't matter. This requires having two antibodies with different epitopes on your antigen. The first antibody is prebound to the plate (preferably to a protein A-coated plate) and is used to capture the antigen from solution. The second antibody is used as the first detection antibody.
In my experience you generally get better sensitivity by filtering the sample over a PVDF-membrane in a 96-well manifold. I once tested this with iodinated Hsc70: 95% of the radioactivity was bound to the membrane. Considering that the desalting column after the iodination reaction will remove most, but not all of the unbound iodine this means that nearly all protein was bound. This will work even with protein in SDS sample buffer. A hydrophobic plate will bind only a few % of the protein, and detergent may interfere. There is also no need for a capture antibody.
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