How are people handling the “validate your protein change by western blot” reviewer request? We already have proteomics data showing protein KD, qPCR showing transcript KD, and a dramatic functional response. I am leaning towards holding my ground not to do western. It is just redundant busy work - ThreadSky

How are people handling the “validate your protein change by western blot” reviewer request? We already have proteomics data showing protein KD, qPCR showing transcript KD, and a dramatic functional response. I am leaning towards holding my ground not to do western. It is just redundant busy work

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pedrobeltrao.bsky.social•8 days ago

Push back for the rest of us :)

samyers-lab.bsky.social•8 days ago

Flight fire with fire.
https://www.biorxiv.org/content/10.1101/2024.04.19.590340v1

msbioworks.bsky.social•7 days ago

mtrost.bsky.social•6 days ago

Show the ratios of all peptides of the protein.

If you have plenty of peptides, and replicates, this can be very convincing.

If it is a protein quantified on few peptides, a WB may make sense.

(Although we recently tested our pipeline and even single hit wonders are amazingly well quantified)

proteomicsnews.bsky.social•6 days ago

metamorpheus.bsky.social•7 days ago

There is an individual for whom I have reviewed several papers. I am quite used to being told by them that my suggestions are beyond the scope, blah blah. Unironically I am satisfied by this non-response because of my tremendous respect for that individual. You may get a + response to your "no".

metamorpheus.bsky.social•8 days ago

benneely.com•8 days ago

You're joking right? I thought those stopped happening 3-5 years back.

metamorpheus.bsky.social•8 days ago

That referee probably works for doge

geremyclair.bsky.social•8 days ago

Here is a good way to waste $$$ but turn a reviewer a mass spec believer. Buy 3 antibodies: the first well validated, and 2 from « less reputable » suppliers. Then show that WB gives you discrepant results. Now point to the reviewer that antibodies are not more reliable than mass spectrometry.

geremyclair.bsky.social•8 days ago

You could also use a typewriter to type your answer and ask the reviewer if he recommends the use of home-casted gels to perform the western blots.

ajbrenes.com•6 days ago

@mehta-lab.com had a paper about this topic that could help in the rebuttal

https://www.nature.com/articles/s41477-022-01314-8

benparkerlab.bsky.social•8 days ago

Yes probably redundant, but there are considerations before pushing back. Do you have lysate remaining or would you have to repeat experiments? How many other ‘battles’ are there in the comments? If there are bigger fights and you have lysate than might be worth a ‘quick’ WB to appease the gods.

dlswaney.bsky.social•8 days ago

I hear you. If this was a well-studied protein with a rock-solid antibody I might consider it. But it’s not. Also, it’s an antiquated double standard to “validate” more specific and quantitative proteomics data with an inferior western blot assay. I would bet the opposite request is far more rare.

ucdproteomics.bsky.social•8 days ago

Tell the reviewers 2005 is calling and wants a word?

paleoproteomicist.bsky.social•8 days ago

I was recently asked to do so on a non-model organism project and I suggested my coauthors say that the proteomics community does not support using Westerns this way and gave them the following pubs: https://www.nature.com/articles/s41477-022-01314-8 and https://www.mcponline.org/article/S1535-9476(20)31024-0/fulltext

mattpadula.bsky.social•7 days ago

Send them my favourite article: https://www.nature.com/articles/d41586-024-03590-0

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