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This modified BCA Protein Assay system greatly increases the sensitivity of protein quantitation. The principle, however, stays the same as the standard bicinchoninic acid assay. Briefly, Cu2+-protein complex formed under alkaline conditions reduces the Cu2+ to Cu1+. BCA binds the Cu+1, resulting in a purple colored complex, which is measured at 562 nm to determine the protein concentration.
Protein + Cu2+ => Cu1+ + BCA => Cu1+- BCA complex
Any protein containing cysteine, cystine, tryptophan, tyrosine, and the peptide bond can reduce Cu2+ to Cu1+.
SPECIAL FEATURES
•Simple and highly sensitive for assay proteins at low concentration.
•Concentration range for protein assay from 1.0-100µg/ml.
•Compatible with many ionic and nonionic detergents.
•Independent of specific amino acid content of proteins.
•Good for assays using cuvettes or a microplate format.
KIT CONTENTS
•Low Protein BCA Reagent A: 125ml
•Low Protein BCA Reagent B: 125ml
•Low Protein BCA Reagent C: 7.5ml
•Protein Standard Solution: 5ml
Advantages and disadvantages using BCA Protein Assay:
This is a very sensitive method for quantitation of proteins at low concentration. As in the standard BCA Protein Assay, the Low Protein BCA Protein Assay is also compatible with many ionic and nonionic detergents at concentrations up to 5%. This assay produces a nearly linear response curve for protein solutions in a concentration range from 1.0µg/ml to 100ug/ml.
Care needs to be taken with protein solutions containing reducing substances or chelating agents. Anything that reduces copper will interfere with the accuracy of the protein quantitation; and reagents that chelate copper will reduce the amount of Cu1+- BCA complex produced during the reaction. If this is the case, Bradford Protein Assay (G1001) should be used.
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.
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