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Thursday, December 1, 2011

Bradford Protein Assay

The Bradford protein assay is a colorimetric assay for the determination of protein concentration, It is dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M. Bradford. The Bradford protein assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive

The principle is based on the shift in absorbance maximum of acidic solution of coomassie brilliant blue dye from 465nm to 595nm when binding to the protein occurs.The colour of the solution turns from red to blue.This dye specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. It should be noted that the assay primarily responds to arginine residues (eight times as much as the other listed residues) so if you have an arginine rich protein, you may need to find a standard that is arginine rich as well. CBBG binds to these residues in the anionic form, which has an absorbance maximum at 595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance maximum at 470 nm (red). The assay is monitored at 595 nm in a spectrophotometer, and thus measures the CBBG complex with the protein.

A standard protein curve can be made using known concentration of BSA and then the test sample protein concentration can be determined.

Bio-Rad has ready to use bradford protein assay dye reagent, bio-rad assay protocol can be found from the below link

http://www.bio-rad.com/LifeScience/pdf/Bulletin_9004.pdf

References

sbio.uct.ac.za/Sbio/documentation/The_Bradford_Assay.doc

http://www.bio-rad.com

www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html

http://www.oardc.ohio-state.edu/stockingerlab/Protocols/BradfordAssay.pdf