I recently did a proteomics experiment on lung cancer cells examining the effect of knocking down my protein of interest, and did a Western blot and qPCR to confirm the knockdown prior to prepping the samples using a TMT-10plex. Upon seeing the proteomics (It was run on two different mass specs. an Orbitrap Fusion Lumos and an Orbitrap QExactive), it appears that protein is not actually knocked down.
By both Western blot and qPCR, levels were 50% and 10% of the controls but by proteomics they were 100% and 50% of the control.
Can anyone shed any light as to why this may be?
I can understand if you don’t want to reveal the knockdown protein, but are you sure you are seeing the right protein via MS? Does it have isoforms, and are you certain you have quantified the correct isoform?
Likewise for the blot which can be notoriously unreliable depending on your antibody. How specific is the antibody and do you know for sure, or are you going by what others have said, or worse still, the manufacturer/supplier information?
It’s the same for qPCR, which I assume would be more specific, but since this isn’t my area, I could be quite wrong.
And what is the control and is it the same in all cases?
Peter G Hains Thanks for the reply, I'm sure the MS is detecting the correct protein because I took the individual peptide sequences and blasted them (They map along the entire protein and there is only one protein coding transcript variant/ isoform).
The antibody used for the Western blot has been extensively validated by our lab and is the first commercially available one we've found that works as well as the homemade one we have been using.
As for the qPCR, the primers I'm using and the antibody used for Western detect opposite termini, so should give a good indication on what's happening along the entire length.
The control here is a non-targeting shRNA and is the same for all three methods.
It is very common for mRNA and protein levels be different. I would say it is rather a norm we see in our work. Monitoring protein synthesis (SILAC approach) and/or turnover kinetics (pulse-chase) might be helpful to understand how quickly levels of this particular protein can be depleted in your knockout/knockdown model once gene is removed or silenced.
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