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Bicinchoninic acid assay
The bicinchoninic acid assay (also known as the BCA assay or Smith assay) is a biochemical assay for determining the total level of protein in a solution, similar to Lowry protein assay, Bradford protein assay or biuret reagent. The total protein concentration is exhibited by a color change of the sample solution from green to purple in proportion to protein concentration, which can then be measured using colorimetric techniques.
Additional recommended knowledge
A stock BCA solution contains the following ingredients in a highly alkaline solution with a pH 11.25:
The BCA assay primarily relies on two reactions.
Firstly, the peptide bonds in protein reduce Cu2+ ions from the cupric sulfate to Cu1+ (a temperature dependant reaction). The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid chelate with each Cu1+ ion, forming a purple-colored product that strongly absorbs light at a wavelength of 562 nm.
At the same time a second (temperature independant) reaction takes place between the reagent and certain side chains (including tyrosine, tryptophan). In order to minimise the effects of this sequence-specific reaction on the assay it is usually carried out at 37 degrees.
The amount of protein present in a solution can be quantified by measuring the absorption spectra and comparing with protein solutions with known concentrations.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Bicinchoninic_acid_assay". A list of authors is available in Wikipedia.|