Linearization of the Bradford protein assay

Orna Ernst*, Tsaffrir Zor

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

244 Scopus citations

Abstract

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.

Original languageEnglish
Article numbere1918
JournalJournal of Visualized Experiments
Issue number38
DOIs
StatePublished - Apr 2010

Keywords

  • Bradford
  • Cellular biology
  • Coomassie brilliant blue
  • Issue 38
  • Protein assay
  • Protein quantification

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