Single amino acids and dipeptides are unaffected in the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue-to-violet complex that absorbs light at 540 nm. Why BCA Protein Assay is Overhyped. Following the incubation, the Folin phenol reagent is added. Another major advantage of the BCA protein assays is the low protein-to-protein variation. The advantage of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the copper solution to allow a one step procedure. For Research Use Only. While the precise mechanism for color formation using the Pierce Modified Lowry Protein Assay is not completely understood, it is known that the reaction occurs as two distinct steps. Figure 6. Both assays were conducted according to the manufacturer’s protocols, in a microplate format. First, protein reacts with alkaline cupric sulfate in the presence of tartrate during a 10-minute incubation at room temperature. Proteins are diverse in their composition and structure, and with some assays the proteins’ differences in amino acid sequence, isoelectric point (pI), secondary structure, and side chains or prosthetic groups result in variation in the colorimetric response. The Pierce BCA Protein Assay produces a linear response curve (R2 > 0.95) and can be performed using two different formats based upon the dynamic range needed to detect the protein concentration of an unknown sample. One cupric ion forms a colored coordination complex with four to six nearby peptides bonds. The BCA Protein Assay is a popular method for colorimetric detection and quantitation of total protein. The Pierce Modified Lowry Protein Assay is based on the colorimetric method introduced by Oliver H. Lowry in 1951. Disadvantages of the Bradford Protein Assay The biggest disadvantage of the Bradford protein assay is that it does not work if detergents or surfactants are in the sample or if the sample is basic… The signal induced by the BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the signal using the biuret reagent. Protein Assay Selection  Protein Assay Technical Handbook. The reduced phosphomolybdic–phosphotungstic acid complex produced by this reaction is intensely blue in color. It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic–phosphotungstic acid complex (the Folin phenol reagent). Accuracy of the Pierce Rapid Gold BCA Protein Assay and Bradford Protein Assay with known protein mixes. 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. Not for use in diagnostic procedures. What is the biggest advantage of using the Bradford protein assay to determine total protein concentration? Figure 7. Figure 5. 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. This colorimetric assay provides the high sensitivity and linearity associated with the BCA assay, but in a fraction of the time it takes to perform a standard BCA assay. This low protein-to-protein variation leads to higher accuracy in determining protein concentration for unknown protein samples. Since the color reaction is not a true endpoint reaction, considerable protocol flexibility is allowed with the Pierce BCA Protein Assay. The standard Pierce BCA Protein Assay detects protein concentrations from 20 to 2,000 μg/mL using a two-component system: Reagent A, a carbonate buffer containing BCA reagent, and Reagent B, a cupric sulfate solution, which are combined to make an apple green–colored working solution that turns purple after 30 minutes at 37°C in the presence of protein. Figure 8. The Pierce BCA Protein Assays combines the well-known reduction of Cu2+ to Cu+ by protein in an alkaline medium (also known as the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid (BCA). Figure 2. Furthermore, the Folin reagent is reactive for only a short period of time after addition. 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.