Protocol

Quantifying Protein by Bicinchoninic Acid

This protocol was adapted from "Techniques," Appendix 2, in Proteins and Proteomics by Richard J. Simpson. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

This protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu⁺ generated under alkaline conditions at sites of complexes between Cu²⁺ and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.