Application Note: In-Situ Microvolume Bicinchoninic Acid Protein Assay

By Peter Brescia, MSc. and Peter Banks, Ph. D., BioTek Instruments, Inc., Winooski, VT

Standard protocols for bicinchoninic acid (BCA) assays using vials or microplates have been modified for use with the Take3™ Multi-Volume Plate suitable for micro-volume analysis. Protein samples and BCA working reagent are added sequentially directly to the microspots of the Take3 plate, incubated, then read on the Epoch™ Multi-Volume Spectrophotometer. Improvements in sensitivity are evident relative to standard protocols using 20:1 volume ratios of BCA working reagent to protein. Comparisons to a "Mini-BCA" assay workflow indicate that in-situ analysis is more accurate.

Bicinchoninic acid (BCA) is one of the most popular reagents for colorimetric protein determinations. Many reagent vendors supply BCA kits for use with cuvette- or microplate-based spectrophotometers. The popularity of BCA stems from ease of use, stability and sensitivity relative to other colorimetric assays. Compared to native protein absorbance at 280 nm, BCA has improved sensitivity and good specificity for protein with minimal interference from nucleic acids, which is always a hazard when quantifying cell lysate or other biological samples. BCA reagents act in conjunction with the biuret reaction to form a purple colored product attributed to the chelation of two molecules of BCA per Cu⁺ ion.