Bicinchoninic acid (BCA) assay in low volume

The BCA assay is a calorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (lambda(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 mu L of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format. (C) 2010 Elsevier Inc. All rights reserved.