TY - JOUR
T1 - Linearization of the Bradford protein assay
AU - Ernst, Orna
AU - Zor, Tsaffrir
PY - 2010/4
Y1 - 2010/4
N2 - Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.
AB - Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.
KW - Bradford
KW - Cellular biology
KW - Coomassie brilliant blue
KW - Issue 38
KW - Protein assay
KW - Protein quantification
UR - http://www.scopus.com/inward/record.url?scp=80355148723&partnerID=8YFLogxK
U2 - 10.3791/1918
DO - 10.3791/1918
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C2 - 20386536
AN - SCOPUS:80355148723
SN - 1940-087X
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 38
M1 - e1918
ER -