From Wikipedia,
the free encyclopedia.
The Bicinchoninic acid assay
or BCA assay is a
biochemical
assay or a
chemical test for detecting
the presence of
protein in a solution, similar
to
Lowry protein assay,
Bradford protein assay or
biuret reagent. It changes
colour from green to purple in
proportion to protein
concentration in a given sample,
which can then be measured using
colorimetric techniques.
Procedure
A stock BCA solution contains 1
g
bicinchoninic acid (BCA), 2 g
sodium carbonate, 0.16 g
sodium tartrate, 0.4 g NaOH,
and 0.95 g
sodium bicarbonate per 100 ml
of aqueous solution. The
pH of the solution should be
11.25 (adjust with
NaOH). The BCA solution is
mixed in 50:1 ratio to 4%
weight/volume
CuSO4*5H2O solution to obtain
a working solution.
In this preparation, 1
ml of the working solution and
20
μl of the protein solution to
be assayed are mixed and incubated
(2h at
room temperature (RT), 30 min
at 37 °C or 15 min at 60 °C and
then cooled to RT.
Kits are also available from
numerous commercial sources.
Typically they are more
concentrated, and assays are
performed in 200 μl volumes within
a
96-well plate for higher
thoroughput.
The BCA assay relies on two
reactions. First, the Cu2+
ions, BCA and the
peptide bonds or some of the
amino acid residues in protein
form a complex which
reduces Cu2+ to Cu+1.
The amount of Cu2+
reduction is proportional to
the amount of protein present in
the solution. Then the Cu+1
ion and BCA form a complex
which has a purple-blue color and
strong absorption at 562
nm. The amount of protein
present in a solution can be
quantified by measuring the
absorption spectra and
comparing with protein solutions
with known concentrations.
References
- P.K. Smith et al. Anal.
Biochem. 150: 76 (1985)
- K. J. Wiechelman et al.
Anal. Biochem. 175: 231 (1988)
- Stoscheck, CM. Quantitation
of Protein. Methods in
Enzymology 182: 50-69 (1990)
