Bicinchoninic acid (BCA) assay in low volume

The BCA assay is a colorimetric 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 (λ_max 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format.