Bicinchoninic acid assay
Encyclopedia
The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith at the Pierce Chemical Company
, is a biochemical
assay
for determining the total level of protein
in a solution (0,5 μg/mL to 1,5 mg/mL), 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.
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The BCA assay primarily relies on two reactions.
Firstly, the peptide bonds in protein reduce
Cu2+ ions from the cupric sulfate to Cu+ (a temperature dependent 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 Cu+ ion
, forming a purple-colored product that strongly absorbs light at a wavelength
of 562 nm.
The bicinchoninic acid Cu+ complex is aided in protein samples by the presence of cysteine, cystine, tyrosine, and tryptophan side chains. At higher temperatures (37oC to 60oC), peptide bonds assist in the formation of the reaction product. Incubating the BCA assay at higher temperatures is recommended as a way to increase assay sensitivity while minimizing the variances caused by unequal amino acid composition.
The amount of protein present in a solution can be quantified by measuring the absorption spectra
and comparing with protein solutions with known concentrations.
Pierce Biotechnology
Rockford, IL-based Pierce Chemical Company was founded in 1948 when Dr. Alan Pierce assumed active management of a company known as Midwest Extraction, which focused on extracting chlorophyll from alfalfa...
, is a biochemical
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
for determining the total level of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
in a solution (0,5 μg/mL to 1,5 mg/mL), similar to Lowry protein assay
Lowry protein assay
The Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. It is...
, Bradford protein assay
Bradford protein assay
The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is subjective, i.e., dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M...
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
Colorimetry
Colorimetry is "the science and technology used to quantify and describe physically the human color perception."It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color perception, most often the CIE 1931 XYZ color space...
techniques.
Mechanism
A stock BCA solution contains the following ingredients in a highly alkaline solution with a pHPH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
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- Bicinchoninic acidBicinchoninic acidBicinchoninic acid is a weak acid composed of two carboxylated quinoline rings.Bicinchoninic acid is most commonly employed by biochemists in the bicinchoninic acid assay, which is used to determine the total level of protein in a solution...
- Sodium carbonateSodium carbonateSodium carbonate , Na2CO3 is a sodium salt of carbonic acid. It most commonly occurs as a crystalline heptahydrate, which readily effloresces to form a white powder, the monohydrate. Sodium carbonate is domestically well-known for its everyday use as a water softener. It can be extracted from the...
- Sodium bicarbonateSodium bicarbonateSodium bicarbonate or sodium hydrogen carbonate is the chemical compound with the formula Na HCO3. Sodium bicarbonate is a white solid that is crystalline but often appears as a fine powder. It has a slightly salty, alkaline taste resembling that of washing soda . The natural mineral form is...
- Sodium tartrateSodium tartrateSodium tartrate is used as an emulsifier and a binding agent in food products such as jellies, margarine, and sausage casings. As a food additive, it is known by the E number E335....
- Cupric sulfate pentahydrate
The BCA assay primarily relies on two reactions.
Firstly, the peptide bonds in protein reduce
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....
Cu2+ ions from the cupric sulfate to Cu+ (a temperature dependent 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 Cu+ ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
, forming a purple-colored product that strongly absorbs light at a wavelength
Color
Color or colour is the visual perceptual property corresponding in humans to the categories called red, green, blue and others. Color derives from the spectrum of light interacting in the eye with the spectral sensitivities of the light receptors...
of 562 nm.
The bicinchoninic acid Cu+ complex is aided in protein samples by the presence of cysteine, cystine, tyrosine, and tryptophan side chains. At higher temperatures (37oC to 60oC), peptide bonds assist in the formation of the reaction product. Incubating the BCA assay at higher temperatures is recommended as a way to increase assay sensitivity while minimizing the variances caused by unequal amino acid composition.
The amount of protein present in a solution can be quantified by measuring the absorption spectra
Absorption spectroscopy
Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of the absorption varies as a...
and comparing with protein solutions with known concentrations.