BCA diluent and standard curve - (Feb/14/2011 )
I have a problem understanding something about the BCA assay. I've tried to read as much as I can online about this, have gone through this forum's archives as well as another bio/methods forum's archives, as well as other people's posted protocols, but I just can't seem to find a concrete answer to one specific thing.
I am extracting protein from mouse brain using a 0.5% SDS lysis buffer (with 150mM NaCl, 50mM Tris, 2mM EDTA, protease & phosphatase inhibitors) and quantifying protein quantity via BCA kit from Pierce, on a microplate. Previously I had been diluting the BSA standard the kit comes with (2mg/mL) in distilled/deionized water for the standard curve, as well as diluting the protein samples to 1:10 in distilled/deionized water. I load 10uL of standards and diluted samples into the wells and combine with 200uL of working reagent.
Pierce’s instructions (as well as other people’s) say to dilute the standards in the same diluent as the samples. Obviously the character of the diluent can change the absorbance of the sample, but does this diluent need to be the lysis buffer that was used on the samples being measured? To get my unknown samples within the standard curve range I dilute them 1:10, and I imagine that the effect of the 0.5% SDS lysis buffer on absorbance is somewhat diminished by being diluted in water, but is it reduced enough to be negligible? I understand that the diluent of the BSA standards should be the same as the samples (whether it be water, lysis buffer, etc), but does it necessarily need to be the lysis buffer that was used to extract the protein (if I am diluting out the unknown protein samples 1:10)? And does everyone actually do this – I mean dilute their standards and protein samples in the lysis buffer they used to get that extract? If I loaded 10uL of standards made with water and 10uL of straight undiluted protein sample there would be a problem, since the unknown protein sample would be 10uL of lysis buffer. But if diluted in water, it is really only 1uL of lysis buffer going into the well.
There was a thread on this in November 2010 where bob1 explained that if you use different diluents you could possibly get different slopes on the standard curves and this is where the problem would be. This is what got me questioning my previous method. I ran standard curves (picture attached) with water, 0.9% NaCl (what the BSA from the kit is diluted in), 0.5% SDS lysis buffer with protease and phosphatase inhibitors, and 0.5% SDS lysis buffer without protease and phosphatase inhibitors (to see if there is an effect). I made both the SDS standards myself (weighing out the BSA); the water and NaCl standards started with the BSA that came with the kit. The water and NaCl curves were very similar, but the SDS slopes were different, and the inhibitors did have an effect. So then it makes sense to use the SDS lysis buffer as the diluent (although I can see a potential flaw with comparing the curves to samples, though won’t get into that otherwise this post will get way too long), but I guess another question is – what do people actually do? The BSA that the kit comes with is not in lysis buffer, so if you used that one of your dilution points would be different. You could of course make up your own solution of BSA in lysis buffer, but then why would the kit come with its own ampule of 2mg/mL BSA?
Sorry for the long post and thank you for reading. Thanks in advance for any insight provided : )
yes, you should include the lysis buffer with your standards, at the same final concentration as your samples. if you can't make the highest standard with buffer then omit it and try to make one as close as possible if you need it.