It is reported that P-glycoprotein (P-gp) can mediate multiple drug resistance. However, it has monomer, dimer and oligomer forms. If I want to detect the change of P-gp expression level in resistant cells by Western blotting, which form better represents the capability of drug efflux?
If you run the protein on SDS-PAGE prior to Western blotting, you should see monomer only because of denaturation of the protein by SDS. In my experience with P-gp, I saw two bands on SDS-PAGE, both quite diffuse, which was attributed to heterogeneous glycosylation. The upper band was not large enough to be dimer. Pre-treating the protein with PNGaseF prior to addition of SDS-PAGE sample buffer should remove the glycosylation, leaving one sharp band, if the digestion is complete.
According to this paper (http://www.ncbi.nlm.nih.gov/pubmed/9374872), P-gp is normally found as a dimer in vivo, at least in some tissues. The amount of monomer seen on Western blot should therefore reflect the total amount of P-gp,which in turn should reflect the efflux capacity.
Dear Dr. Shapiro,
I saw three bands in my SDS-PAGE results, just like the figure shown in Abcam's website for ab89322 (http://www.abcam.com/p-glycoprotein-antibody-ab98322.html).
The largest band did not reach 170 kD, I guess it might due to cell line difference.
So far I want use the ~140 kD band to quantify the expression of P-gp, and I am currently trying another P-gp antibody at this moment.
Thank you! The paper you provided is very useful.
The Western blot on the web page showed a band at ~140 kDa, which is full-length unglycosylated P-gp, and two bands of lower molecular weight that are either proteolytic fragments or cross-reactivities. If you look at the Western blots on the same web page for other anti-P-gp antibodies, including ab170903 and ab170904, you will see better results.
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