I have read an article in which the author mentioned that the cell lysates were mixed with the primary antibody before loading into the gel, then transferred to the membrane and probed with the secondary antibody. I'm wondering if I can do the similar experiment with patients' sera. Thank you.
You may get some problems in your analysis. There is an incubation step with blocking buffer that precedes the primary antibody incubation. This step avoids unspecific tags. In addition, it would be necessary to know the molecular weight of the protein of interest and the antibody, because with the binding of the primary antibody, the molecular weight will increase and other bands that are not of interest may appear. Another important point is to know the quality of your antibody and whether it is monoclonal or polyclonal.
Hi Chit,
I do not know the precise specifications of the method you mentioned. However, the primary problem is that you need to run your WB under absolutely non-denaturing conditions. As soon as you add SDS, DTT, or BME to your sample (or gel or running buffer) and/ or boil it, you will abolish the antibody binding and its integrity (as it will dissociate in its heavy and light chains). If you maintain the undenatured conditions and, maybe, additionally stabilize the binding by crosslinking the antibody to the target, this approach might work.
I concur with the points raised by Azuil.
From my perspective, I do not see the advantage of this strategy in a typical immunoassay like a WB detection.
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