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Life Science Applications
Pierce BCA
Thermo Scientific Evolution 200 Series User Guide 179
Overview
Related Topics
Application Settings
Working With Standard Curves
Making Pierce BCA Measurements
BCA Kits and Protocols
Commercial BCA Protein kit manufacturers typically outline procedures for two different
protein concentration ranges:
• A regular assay uses a 20:1 reagent/sample volume ratio.
• A mini assay uses a 1:1 reagent/sample volume ratio. To prepare sufficient volume of these
1:1 mixtures for cuvette measurements, use a sample volume of at least 3 ml and 3 ml of
BCA reagent.
Follow the assay kit manufacturer’s recommendations for all standards and samples
(unknowns). Ensure each is subjected to the same timing and temperature throughout the
assay.
Protein standards (BSA) for generating a standard curve may also be provided by the
manufacturer for the BCA assay.
The BCA Protein Assay, which was patented by Pierce
Biotechnology, part of Thermo Fisher Scientific, remains a
cornerstone for accurately measuring protein
concentration in biological samples. The method uses
bicinchoninic acid (BCA) as the detection reagent for
Cu
+
, which is formed when Cu
2+
is reduced by protein in
an alkaline environment. A purple-colored reaction
product is formed by the chelation of two molecules of
BCA with one cuprous ion (Cu
+
). The resulting Cu-BCA
chelate formed in the presence of protein is measured at
562 nm. While this assay is linear over a wide
concentration range, the calibration curves are best
represented as second order. Preformulated reagents of
BCA and CuSO
4
are available in kit form from us.
Note If running the assay at 60 °C, doubling the volumes may afford greater insurance
against skewed results from evaporation/condensation within the sealed reaction tube.
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