Hi all,

I'm running into a serious problem with quantifying my protein samples. We're using a typical lysis buffer (tris/glycerol/1%sds), but are running into issues where our lysis buffer blanks are giving higher absorbance readings than our actual protein samples. What we typically do is run the albumin standards from pierce along with our protein samples and the lysis blanks. Subtract the lysis blank values from our protein sample absorbances and then compare the resulting value to the standard curve of albumin.

I'm not quite sure what to make of this because the assay (using the same parameters) used to work just fine for us in the past. We've tried loading just a set amount of protein (10 ul) onto SDS-page and there is definately protein there. Has anyone run into a similar issue and been able to troubleshoot what the root cause was?

Thanks

Do you have EDTA, beta-mercaptoethanol in your lysis buffer? These are the most serious interferring substances for that assay.

Also, when you said that you used blank with lysis buffer, did you used the same amount as sample volume to correct the assay? Use diluted sample can help minimize the interferring effects.

Maybe you should check every compound of your lysis buffer seperately... when the blanks are that high, it seems like there is something in it... especially when it worked in former assays.

Interestingly enough, the glycerol was the component which had the strongest reaction to the BCA kit turning dark purple almost immediately after the addition of the working reagent. Is there something I could substitute? Currently we use a 1% SDS, .06 M Tris-HCL, 10% glycerol lysis buffer.