Does anyone know what the best way to quantify PD-L1 expression is? Following treatment, I see a massive PD-L1 upregulation in mRNA level using qPCR and but only a partial upregulation using flow cytometry (done without permeabilizing- surface expression only). However, I see no difference in expression using a western blot (using PD-L1 (E1L3N®) XP® Rabbit mAb #13684). Has anyone come across similar issues? Any ideas on how PD-L1 is recruited to the surface?
Many thanks
Hi Ashwin,
Western blot gives better results in term of checking protein expression level.
Have you normalized your protein expression to a internal control like Actin?
Not always mRNA levels represent protein levels. There are post-transcription and post-translation could affect a protein level in a cell.
Good luck
Would trust your WB data more since the size criterion adds one more level of stringency; does your WB data match well the predicted size (CellSignaling has some supporting evidence on that one)? However, theoretically possible that the protein detected by WB is not the predicted one (knock-down controls always good to have, for both techniques). Not clear why your "massive" mRNA upregulation is not accompanied by protein upregulation, but there could be a number of reasons for that relating to tight post-transcriptional regulation.
Good luck!
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