BCA test

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Bicinchoninic acid
The purple Cu + -bicinchoninic acid complex

The BCA test is a biochemical method to quantify proteins using bicinchoninic acid (BCA) .

principle

The BCA test was published in 1985 by PK Smith and colleagues. It is based on the Lowry test of 1951, which in turn draws on the biuret reaction for protein determination from 1949 and the Folin-Ciocalteu reagent from 1927. The BCA replaces the Folin-Ciocalteu reagent in the Lowry test in the BCA test.

Bivalent copper ions bind to the peptide bond and are reduced to monovalent copper ions at an alkaline pH . In the presence of BCA, the copper ions form a blue-violet dye complex with BCA. Bicinchoninic acid is stable in an alkaline pH value, which means that, in contrast to the Lowry test, one less step is necessary. The detection limit is comparable to the Lowry test. Furthermore, the BCA test is less susceptible to interference with the chaotropic urea and guanidinium chloride , with the salts ammonium sulfate and cesium hydrogen carbonate and with the surfactants Triton X-100 , SDS , Brij 35 , Lubrol , CHAPS , CHAPSO and octylglucoside .

The reagent solution consists of the stock solutions A (10 g / L sodium bicinchoninate, 20 g / L sodium carbonate , 1.6 g / L sodium tartrate , 4 g / L sodium hydroxide , 9.5 g / L sodium hydrogen carbonate , to a pH value of 11, 25 adjusted with sodium hydroxide) and B (40 g / L copper sulfate pentahydrate) in a mixing ratio of 50 to 1. The reagent solution is freshly prepared from the stock solutions before use. The sample is diluted 1:50 with the reagent solution and heated for 30 minutes at 60 ° C. The resulting dye is quantified by photometry at a wavelength of 562 nm. While the amino acids cysteine , cystine , tryptophan and tyrosine in particular reduce divalent copper ions at room temperature, the copper ions are also reduced at 60 ° C by the peptide bond, which means that the measurement result is less dependent on the amino acid composition of the proteins being measured.

literature

  • CM Stoscheck: Quantitation of protein. In: Methods in enzymology. Vol. 182, 1990, pp. 50-68, PMID 2314256 .

Individual evidence

  1. ↑ PK Smith, RI Krohn, GT Hermanson, AK Mallia, FH Gartner, MD Provenzano, EK Fujimoto, NM Goeke, BJ Olson, DC Klenk: Measurement of protein using bicinchoninic acid. In: Analytical biochemistry. Volume 150, Number 1, October 1985, pp. 76-85, PMID 3843705 . doi : 10.1016 / 0003-2697 (85) 90442-7 .
  2. ↑ Oliver H. Lowry et al. : Protein measurement with the Folin phenol reagent. In: J. Biol. Chem. Volume 193, No. 1, 1951, pp. 265-275. PMID 14907713 . PDF .
  3. ^ AG Gornall, SJ Bardawill, MM David: Determination of serum proteins by means of the biuret reaction. In: J Biol Chem. Volume 177 (2), 1949, pp. 751-766. PMID 18110453 .
  4. ↑ Folin, O., and Ciocalteu, V .: On tyrosine and tryptophane determinations in proteins . In: Journal of Biological Chemistry . 73, 1927, p. 627.
  5. ↑ a b Olsen BJ, Markwell J: Assays for the Determination of Protein Concentration . In: Current Protocols in Protein Science . 2007, pp. 14-17.
  6. ^ A b c John M. Walker: The Protein Protocols Handbook. Springer Science & Business Media, 2008, ISBN 978-1-603-27259-9 , p. 11.
  7. ^ A b c d Alfred Pingoud : Working methods of biochemistry. Walter de Gruyter, 1997, ISBN 978-3-110-16513-5 , p. 152.