Sunday, November 28, 2021

Affinity Enrichment is nearly always better than (attempts at) Affinity Purification!

This paper is older, but if I send the same link to a study to three people in under 2 weeks maybe it's good to talk about it again! 

Biologists do tons of "pull-down", "immunoprecipitation", "immunoenrichment" things from the simple to the "number of steps that don't even seem remotely possible to pull off but is for some reason absolutely acquired to find this target". 

Despite how they set up the experiment, there is something for them to learn from this paper, particularly if they haven't done an experiment like this in 20 years and are expecting a short list of presence/absence. Now, I'm much more likely these days to send out a report from FragPipe for quantitation, because it now has match between runs and match between runs FDR and I can have a saved template for "so and sos triple knock out mutant IP" because I'll always forget at least one setting in MaxQuant from the last time I ran it, but the principle is the same. Simpler + better semi-quantitative = better. 

That is the first paper I send them. 

The second thing I send them is from a great little proteomics core lab at Colorado State University. Here is a direct download link for this PDF. It's called "Detergents in Mass Spectrometry" and it's important. 

Definitely definitely definitely keep this in mind -- A few years ago some biologist got the great idea to start washing IPs, etc., of detergent used to lyse the cells with the exact same detergents used to lyse the cells. I believe this to be the most popular idea in all of biology of the last 40 years because EVERYONE does this now. The uptake from isolated occurrences to ubiquitous has been mindblowing. I don't think PCR caught on as fast as washing detergent out of things with detergent. As far as I can tell, every person that I've been able to catch in the act (or....after I've spent loads of time removing Triton X-100 or NP-40 from an HPLC....) has experienced zero ill effects from just removing the detergent from their wash solution and following that protocol to the letter. 

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