Abstract
The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid. The reduction of copper is mainly caused by four amino acid residues including cysteine or cystine, tyrosine, and tryptophan that are present in protein molecules. However, unlike the Coomassie dye-binding methods, the universal peptide backbone also contributes to color formation, helping to minimize variability caused by protein compositional differences. Compared to the Bradford assay, the BCA assay is more objective since the universal peptide backbone also contributes to color formation. One disadvantage of the BCA assay compared to the Bradford assay is that it is susceptible to interference by some chemicals present in protein samples, including reducing agents (i.e., DTT and beta—mercaptoethanol), copper chelators (i.e., EDTA, EGTA) and buffers with high concentration, which can be avoided by generating diluted samples.
Keywords: Protein concentration, Protein measument, BSA standards, Bradford assay, Reduction of copper
Materials and Reagents
Equipment
Procedure
Acknowledgments
This work was done in the Andrew Binns Lab in the Department of Biology at University of Pennsylvania, USA and supported by National Science Foundation grants MCB 0421885 and IOS-0818613.
References
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hi fanglian , i am under taking a research project which involves the evaluation of protein membrane vesicles in erythrocytes , would the same principle apply to my blood sample as in i have to make a several dilution factors ??
Hi Maria,Would you lease put your question in English? Thanks.
I need to determine how much protein I have in my sample;How much sample need to use this method or as both of my sample used in this method?I'm not good in english sorry, but is important for me know that both of my sample can process this method if I have only 1mg can used this method.I explain?
Hi, Maria,My apologies for the late response. To determine how much sample is used for BCA protein assay, you should first make several dilutions of your sample, like 1:10, 1:20, 1:50... (if your sample is limited, you can start with a higher dilution. The absorbance readings (at 562 nm) which fall into the linear section of the standard curve can be used to calculate the concentration of your undiluted protein sample.Hope my answer helps. Please let me know if anything is unclear to you.Good luck,Fanglian