Bicinchoninic acid assay: Difference between revisions
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The '''bicinchoninic acid assay''' ('''BCA assay'''), also known as the '''Smith assay''', after its inventor, Paul K. Smith at the [[Pierce Biotechnology|Pierce Chemical Company]],<ref>{{cite journal | author=Smith, P.K., ''et al.'' | title=Measurement of protein using bicinchoninic acid | journal=Anal. Biochem. | year= |
The '''bicinchoninic acid assay''' ('''BCA assay'''), also known as the '''Smith assay''', after its inventor, Paul K. Smith at the [[Pierce Biotechnology|Pierce Chemical Company]],<ref>{{cite journal | author=Smith, P.K., ''et al.'' | title=Measurement of protein using bicinchoninic acid | journal=Anal. Biochem. | year=19856–85 | doi=10.1016/0003-2697(85)90442-7 | pmid=3843705 | issue=1}}</ref> is a [[biochemistry|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 [[colorimetry|colorimetric]] techniques. |
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==Mechanism== |
==Mechanism== |
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Revision as of 06:13, 4 October 2011
The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith at the Pierce Chemical Company,[1] 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.
Mechanism
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 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 Cu1+ ion, forming a purple-colored product that strongly absorbs light at a wavelength of 562 nm.
The bicinchoninic acid Cu1+ 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.[2]
The amount of protein present in a solution can be quantified by measuring the absorption spectra and comparing with protein solutions with known concentrations.
See also
References
- ^ Smith, P.K.; et al. (19856–85). "Measurement of protein using bicinchoninic acid". Anal. Biochem. (1). doi:10.1016/0003-2697(85)90442-7. PMID 3843705.
{{cite journal}}: Check date values in:|year=(help); Explicit use of et al. in:|author=(help)CS1 maint: year (link) - ^ Olsen BJ and Markwell J. (2007). "Assays for the Determination of Protein Concentration" (PDF). Current Protocols in Protein Science: 14–17.
{{cite journal}}: Unknown parameter|supplement=ignored (help)
- Wiechelman, K., Braun, R. and Fitzpatrick, J. (1988). "Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation". Anal. Biochem. 175 (1): 231–7. doi:10.1016/0003-2697(88)90383-1. PMID 3245570.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - Stoscheck, CM. (1990). "Quantitation of Protein". Methods in Enzymology. 182: 50–69. doi:10.1016/0076-6879(90)82008-P. PMID 2314256.