Thermo Scientific Pierce Protein Assay Technical Handbook Version 2

BCA-based Protein Assays
Bicinchoninic Acid (BCA)-based Protein Assays
In 1985, Paul K. Smith, et al. introduced the BCA Protein
Assay. Since then it has become the most popular method
for colorimetric detection and quantitation of total protein.
The BCA Protein Assay has a unique advantage over the
Modified Lowry Protein Assay and any of the Coomassie dyebased
assays – it is compatible with samples that contain up
to 5% surfactants (detergents).
Briefly, the sample is added to the tube or plate containing the
prepared BCA Working Reagent and after a 30-minute incubation
at 37°C and cooling to room temperature, the resultant purple
color is measured at 562nm. The protocol is similar for the Micro
BCA Protein Assay, except the ratio of sample volume to working
reagent is different and the tubes are incubated for 60 minutes
at 60°C.
Chemistry of BCA-based Protein Assays
The BCA Protein Assay combines the well-known reduction
of Cu 2+ to Cu 1+ by protein in an alkaline medium with the highly
sensitive and selective colorimetric detection of the cuprous
cation (Cu 1+ ) by bicinchoninic acid (Figure 1). The first step is the
chelation of copper with protein in an alkaline environment to
form a blue colored complex. In this reaction, known as the biuret
reaction, peptides containing three or more amino acid residues
form a colored chelate complex with cupric ions in an alkaline
environment containing sodium potassium tartrate. This became
known as the biuret reaction because a similar complex forms
with the organic compound biuret (NH 2 -CO-NH-CO-NH 2 ) and the
cupric ion. Biuret, a product of excess urea and heat, reacts with
copper to form a light blue tetradentate complex (Figure 2). Single
amino acids and dipeptides do not give the biuret reaction, but tripeptides
and larger polypeptides or proteins will react to produce
the light blue to violet complex that absorbs light at 540nm. One
cupric ion forms a colored coordination complex with four to six
nearby peptides bonds.
The intensity of the color produced is proportional to the number
of peptide bonds participating in the reaction. Thus, the biuret
reaction is the basis for a simple and rapid colorimetric reagent
of the same name for quantitatively determining total protein concentration.
Since the working range for the biuret assay is from
5 to 160mg/mL, the biuret assay is used in clinical laboratories for
the quantitation of total protein in serum.
Step 1.
Step 2.
Protein + Cu 2+ 0H
Cu 2+
- 00C
N N
Cu 1 + + 2BCA Cu 1 +
- 00C
N BCA
Cu 1 +
N
Complex
COO -
COO -
Figure 1. Reaction schematic for the bicinchoninic acid (BCA)-containing
protein assay.
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.
15