One disadvantage of the Lowry method is that many common substances, such as K +, Mg 2 +, NH 4+, EDTA, Tris-HCl, carbohydrates, and reducing agents, interfere with the method. A53225,A53226,23225,23227,23221,23228,23223,23222,23224,23230, Manage instrument use, information, and service, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Chromatography Columns, Resins, & Spin Filters, BCA Protein Assay - Reducing Agent Compatible, User Guide: Pierce Rapid Gold BCA Protein Assay Kit, User Guide: Pierce BCA Protein Assay Kit − Reducing Agent Compatible, User Guide: Modified Lowry Protein Assay Kit, Application note: A rapid, sensitive protein assay for the accurate analysis of protein concentrations, Tech Tip: Determine acceptable wavelengths for measuring protein assays, Tech Tip: Eliminate interfering substances from samples for BCA Protein Assay, Tech Tip: How to use a protein assay standard curve, Tech Tip: Protein assay compatibility table, Less protein:protein variation than Coomassie dye methods, Significantly less (14–23%) protein:protein variation than Coomassie dye methods, Less protein:protein variation than BCA, Coomassie dye, or Lowry methods, Reducing reagents, Chelators, detergents, tris, tricine, glycerol. Disadvantages: Basic conditions and detergents, such as SDS, can interfere with the dye’s ability to bind to the protein; however, there are detergent-compatible Bradford reagents.
Also, like the absorbance at 280 nm technique, the Bradford assay depends on … The BCA assay is highly sensitive and has a linear range of 20-2,000µg/ml. Since the color reaction is not a true endpoint reaction, considerable protocol flexibility is allowed with the Pierce BCA Protein Assay.
Figure 8. Figure 1. The reduced phosphomolybdic–phosphotungstic acid complex produced by this reaction is intensely blue in color. Figure 3. BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. (B) The same method described in (A) was used to generate standard curves for the Pierce Rapid Gold BCA Protein Assay and Bio-Rad Bradford Protein Assay. Figure 2. In this reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate. The BCA Protein Assay is a popular method for colorimetric detection and quantitation of total protein. In the second step of the color development reaction, the Pierce Rapid Gold BCA chelator reacts with the reduced (cuprous) cation (Cu+) that was formed in step one to produce an intensely gold-colored reaction product. Figure 7. In the second step, BCA reagent, a highly sensitive and selective colorimetric detection reagent, reacts with the cuprous cation (Cu+) that was formed in the first step and produces a purple-color. (A) Purified BSA in 0.9% saline (0–2 mg/mL) was used to generate standard curves for the Pierce Rapid Gold BCA Protein Assay and the standard BCA assay. Another major advantage of the BCA protein assays is the low protein-to-protein variation. First, protein reacts with alkaline cupric sulfate in the presence of tartrate during a 10-minute incubation at room temperature. Standard curves for protein quantitation assays. The copper complex exhibits a strong linear absorbance at 480 nm with increasing protein concentrations.