I am designing an antibody to detect the protein LynA in western blot. Right now I have 8 potential antibodies (SCFVs) that may or may not bind by western blot. I have an apparatus that lets me probe a nitrocellulose membrane that has lynA attached to it with all of the 8 antibodies in parallel and run a control antibody that I know binds for reference. See the attached image for the result of this assay. Each vertical bar represents a narrow well to which I added one antibody, either mine or a commercial antibody I know will bind. I can clearly see that the commercial antibody has bound to the protein. However, My antibodies show a color change at that position too. The area where I would expect the protein to be is a lighter color in wells that have been probed with my SCFVs. Is this lighter color an indication of binding to the target? What else could account for this color change?
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