We've been using a new method for protein extraction (QIAGEN's Qproteome FFPE tissue) in our lab but weren't getting good results in western blots. I've realised that the extraction method includes the use of mercaptoethanol, so its reducing effect must be producing artifactual readings in quantification (we normally use BCA assays for protein quantification), so we can't prepare adequate samples for PAGE.
I'd rather avoid having to set up a new quantification method in the lab, so am considering other options to circumvent the issue. I'd figured out I could try precipitating the protein, discarding supernatant and resuspending to try and get rid of the mercaptoethanol in the sample.
Besides this, I was wondering if anyone would have any suggestions for compounds that I could use to block that reducing effect. Thermo have a "Reducing Agent Compatible" BCA kit, but obviously don't specify what this "compatibility agent" is. Any advice would be much appreciated.
Thanks!
There is a factor which may influnce the results (no idea to what degree tho). Mercaptoethanol tends to form abducts with free cysteines so just to discard that, I would use DTT instead, if you still get bad results, then it's definitely the reducing agent aspect. TCEP is a more exotic reducing agent that has the advantage that it doesn't regenerate like sulfides do, so in theory, unless an excess is used, all the reducing agent should be gone once the disulfide bonds are reduced.
The Bradford assay is another easy-to-use protein assay that is not affected by reducing agents. It suffers interference from detergents, however, so if your samples have a lot of detergent then the Bradford may not work.
TCA precipitation of protein from samples is a good way to get rid of interfering substances such as mercaptoethanol, if you do want to use the BCA assay.
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