Hi,
I've performed a protein extraction on different insect tissues using a 2D DIGE lysis buffer (containing CHAPS, ureum, thioureum and DTT). When performing a BCA assay, my samples (which often have a bit of a colour) become purple instantly when I add the reagent. So eventually I have to make very diluted samples ( 1/20.000) in order to be in range for the BCA assay.
When I then perform WB using 20 and 50 µg of protein (based on the BCA results), I never see a signal. It seems to me that the lysis buffer interferes with the BCA assay.
Is this possible? If so, what other buffers (for example, RIPA or PBS?) can I use to perform protein extraction that are compatible with BCA?
Thanks a lot.
a) The pH of your sample matrix may be wrong and b) the sample buffer concentration may be too high. Wrong pH is usually not a problem, but if the buffering capacity is too high, it will overwhelm the reagent solutions' pH.
Another possibility: do you have a significant concentration of metal ions in your sample?
Dear STijn
i think that the problem is the presence of DTT in your buffer.
In fact BCA is not compatible with DTT and other reducing agents, because it involves the reduction of copper by proteins in an alkaline medium (biuret reaction) to produce sensitive and selective colorimetric detection by a copper chelator (BCA)
and in presence of reducing agent the copper is directly reduced by the reducing agent.
So i think that if you need the presence of the reducing agent for the lysis or you change the method for protein quantification (eg. Bradford) or do you remove the DTT after extraction by performing or desalting or dialsys.
good luck
Manuele
Yes, the DTT - completely overlooked this in the post.
You have to get rid of it before the Ni-NTA.
I am not 100% sure, but I think the urea, and potentially the thiourea, react directly with the reagent.
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