Hello,
Does anyone have experience with PD-L1 detection by SDS-PAGE? I am using 2 antibodies from Cell signaling Technology and E-bioscience with apposite outcomes. Furthermore, when reprobing the membrane and re-detecting the PD-L1 antibody, I get a different result and a different binding profile. I was wondering what can be the science behind it.
Thank you.
I am not an expert at this, but I do know that antibodies are developed against particular regions of the protein. So one antibody may have been developed against the N-terminus and another against the C-terminus. Maybe the C-terminus is typically bound to something and so the N-terminus is more free to binding the antibody.
Is there any way that you could use a negative control like silence the expression of PD-L1 or use a cell line and hope your antibodies don't detect PD-L1?
Dear Hamadi,
the most likely answer is that the method protocol should be revised. A common source of uncertainty comes from the experimentator self as well as from the tools and utilized materials. In this context, it is also possible that the antibodies used are not specific. For reference (and the science behind it) see e.g. Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 2015;521(7552):274-6. doi: 10.1038/521274a).
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