I am trying to quantify my protein from cell samples that I lysed with Cell Signaling's 10X Lysis buffer (#9803) that I supplemented with cOmplete protease inhibitor tablets (EDTA-free, Roche 11836170001). When I used a Bradford Protein Assay kit from Genesee (Cat #: 18-443), the blank using this lysis buffer reacted and turned blue as well.
Is there any reason why the bradford kit would react with this lysis buffer/protease inhibitor? Are there other kits that would work for measuring protein concentration in samples that were lysed with these reagents?
Thanks!
The lysis buffer may contain some substance that is incompatible with the Bradford assay. An example would be a detergent. You can see a Bradford reagent compatibility chart here:
www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6852.pdf
Hi
I agree with Adam
Most detergents reacts with assay
Try to dilute your sample 1:20 or 1:50
Also, do your BSA Standard curve in the same Lysis Buffer 1:20 or 1:50 diluted as your sample = take 1mL Buffer add 19mL H2O and use it as a diluent for BSA
Don`t forget to add Blank reduction to your Std. curve and your sample.
Good luck
Dear @Catie
from the company web site it seems that the buffer that you use contain 1% of tryton x-100 and it is tio high.
you can try to test several buffer dilution until you do not obtain a abs of the blank comparabile o sligty higher ( eg. 0.1 higher) that a blank performed by pbs and repeat you determination using this sample dilution of course il will work only if your extracts are highly concentrated.
good luck
manuele
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