Hi, I use BCA determine protein content in mice colon tissue sample prepared using RIPA buffer supplemented with protease and PMSF. I suspect that the BCA highly over estimated the protein content. It showed my tissue lysate has 80 mg/mL protein. But when I do the downstream western blot, and stained. Only non-diluted sample well showed a little shadow of band (lane 4 and 5).
I also did try using above complete tissue buffer as blank in BCA well. And it gives a super super purple color that so high and can not be measured.
Do anyone know what's the reason? and do you have other protein determination method recommended? Thank you
It sounds like something in your RIPA buffer is interfering with the BCA assay. What is in the buffer?
That does seem quite high for protein concentration. You could always try a Bradford, but I would check to make sure your protocol for BCA is correct first.
Did you probe for any loading controls (e.g. actin, tubulin, GAPDH)? Another way to see how much protein you have is by staining your gel directly with a Coomassie blue stain (Just soak the gel in the Coomasie blue and leave it for ~1/2 hr and then rinse it off (either with water or a destaining solution) to see the lanes). I'm not sure how you're expecting to see bands with just your PVDF membrane. How does the actual film look (or the scan if you're using fluorescent secondaries)?
Please refer to this, someone has encountered the same problem as you.
https://www.researchgate.net/post/Bio-rad_protein_assay_reagent_changes_color_in_RIPA_buffer
Basically, Bio-Rad assay is a Bradford assay and it is well known and documented that detergents (as well as many other chemicals) can cause high backgrounds.
Bio-rad protein assay reagent changes color in RIPA buffer?. Available from: https://www.researchgate.net/post/Bio-rad_protein_assay_reagent_changes_color_in_RIPA_buffer [accessed Apr 16, 2017].
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