I was doing an over-expression experiment of a gene for a year. It is a surface protein. The biggest trouble is that I can never detect the gene overexpression by western blot. Interestingly, when I stain it with a flow cytometry antibody made recently (I also validate the antibody myself), there is actually a great amount of surface protein detected. Why can't the surface protein be detected by WB? Is the same reason that people detect something like CTLA-4, PD-1 by flow cytometry instead of western blot?
Probably because the antibody that you are using detects the target protein in its native confirmation and not the denatured form. That's why you get the signal in FACS when the protein is in native confirmation and no signal in the Western blot where the proteins get denatured.
You may use a different antibody that recognizes a denatured form of your target protein.
High molecular weight proteins may be easier to refold back to its native form even after immobalized in the PVDF/nitrocellulose membrane. You have to make sure that the membrane is always in the buffer and not dried up accidentally throughout the entire western process.
If your antibody works for FACS and WB maybe you need run a WB under condition not denatured
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