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The principle of the bicinchoninic acid (BCA) assay is similar to the Lowry procedure, in that both rely on the formation of a Cu2+-protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to the protein present. In the second step, BCA forms a purple colored complex with Cu+1, which is detectable at 562 nm.
Protein + Cu2+ => Cu1+ + BCA => Cu1+- BCA complex
It has been shown that cysteine, cystosine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2+ to Cu1+. BCA forms a purple-blue complex with Cu1+ in alkaline environments, thus providing a basis to monitor the reduction of alkaline Cu2+ by proteins at absorbance maximum 562 nm.
SPECIAL FEATURES
•Simple and sensitive in protein assays.
•Faster and easier than Lowry protein assay.
•Compatible with many ionic and nonionic detergents.
•Independent of specific amino acid content of proteins.
•Accurate over a wide range of protein concentrations.
KIT CONTENTS
•BCA Protein Assay Reagent 1: 750ml
•BCA Protein Assay Reagent 2: 25ml
A special note for using BCA Protein Assay:
BCA Protein Assayis compatible with many ionic and nonionic detergents at levels up to 5%. BCA Protein Assay is also less complicated to perform than the Lowry Protein Assay . In addition, it produces a nearly linear response curve across a wide range of protein concentrations. The standard BCA Protein Assay detects protein from 20-2,000 µg/ml. The Low Protein BCA Assay (G1003) has a narrower dynamic range of 1-100 µg/ml.
Care needs to be taken with protein solutions containing reducing substances or chelating reagents. Substances that reduce copper will interfere with the accuracy of the protein quantitation; and reagents that chelate copper will reduce the amount of Cu1+- BCA complex formed during the reaction. When this is the case, use the Bradford Protein Assay (G1001).
REFERENCES
•Smith, PK, et al., 1985, Measurement of protein using bicinchoninic acid. Anal Biochem. Oct;150(1):76-85.
•Stoscheck, C. 1990,Quantification of Protein. Methods in Enzymology, 182:50-68.
•Lowry, O., et al., 1951, Protein measurement with the Folin phenol reagent. J. Biol.Chem.193:265-275.
•Protein Standard Solution: 10ml
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