Hi there,
I recently lysed some tissue samples but found my lysis buffer interfered with the bca protein assay so my protein concs were not accurate. The buffer I used (0.01M PBS pH 7.4, 20% glycerol + protease inhibitors) was one someone in our lab previously used though this person did not measure protein conc. I've determined that the glycerol interfered with the assay.
I'm planning on swithing to RIPA buffer but was wondering if there is way to salvage my already lysed tissues. Do I simply dilute with RIPA till the glycerol intereference is lost or do I have to go down the elute through a column and dialyse route?
Any advice would be greatly appreciated.
Many thanks.
Hi David,
According to the BCA compatibility table, BCA assay can be used on samples containing up to 10% glycerol. So you shouldn't have a big problem. I would suggest you to dilute a small amount of your tissue lysates that you are going to use for the BCA assay till you are below these 10% and measure protein concentration. The rest of your samples can be kept in the original lysis buffer, if it's compatible with other assays you are planing to perform on your samples.
Good Luck
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
You could also see if there is a protein assay available that supports that high glycerol, but I do not know for sure. I typically dilute my tissue samples in detergent buffers to 1/5 before determination of the protein with detergent compatible assays. If you have a high protein concentration that still fits nicely to the standard curve of the assay after dilution, I would go with the dilution like Natalia already noted.
I might try acetone precipitating the proteins out then resuspend in RIPA buffer. I've done it before with acyl rac experiments and it hadn't occurred to me I could do the same with those samples. Many thanks for the advice guys!
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