BCA Protein Assay

This experiment describes the adaptation of the bicinchoninic acid (BCA) protein assay to a flow injection analysis. The assay is based on the reduction of Cu + to Cu+ by the protein, followed by the reaction of Cu+ with bicinchoninic acid to form a purple complex that absorbs at 562 nm. Directions are provided for the analysis of bovine serum albumin and rabbit immunoglobulin G, and suggestions are provided for additional analyses. 

Determine the amount of adsorbed protein on the particles by using a suitable protein assay technique, such as the bifunctional chelating agents (BCA) Protein Assay (Thermo Fisher). 

Figure B1.1.4 Graph of the color response curves obtained with Pierce s BCA Protein Assay Reagent using bovine serum albumin (BSA) and bovine gamma globulin (BGG). The standard tube protocol was performed and the color was measured at 562 nm in a Hitachi U-2000 spectrophotometer.

Preformulated versions of the BCA reagent are now commercially available from Pierce (BCA Protein Assay Reagent) or Sigma (Bicinchoninic Acid Kit for Protein Determination). This assay can be performed in test tubes or microtiter plates (see Alternate Protocol 4), and has a working range of 20 to 2000 pg/ml. 

BCA Protein Assay Reagent Kit (Pierce) or Bicinchoninic Acid Kit (Sigma) containing ... 

The protein-to-protein variation in the amount of color produced with the BCA Protein Assay Reagent (CV = 15% for the group of 14 proteins at 1000 pg/ml in the standard tube protocol) is similar to that observed for the modified Lowry protein assay reagent. 

Since the BCA protein assay is not a true end-point assay, the amount of color produced varies with the incubation time and the incubation temperature. While this allows considerable flexibility in optimizing the BCA assay for each application, it also requires that the optimized procedure be followed exactly every time the assay is done. 

Figure Bl.1.4 shows the color response curves obtained with the BCA Protein Assay Reagent using BSA and BGG. The graph shows the net absorbance at 562 nm versus the protein concentration at seven concentrations from 125 to 2000 pg/ml for each protein. Note that the response curve for BGG is higher than the response curve for BSA.

Plus Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be underestimated by -40%. (From Table B1.1. 5, the response ratio for IgG is -0.58 for IgG compared to 1.00 for BSA.) If the BCA Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be overestimated by -15%. (From Table B1.1.5, the response ratio for IgG is -1.15 for IgG compared to 1.00 for BSA.) On the other hand, if BGG had been used for both standard curves, the total protein estimates for the sample would have been in much greater agreement between the two methods. 

Use of the BCA protein assay in a microtiler-plate format which utilizes a microwave oven as the heat source to shorten the color development time to 20 sec. 

Describes a procedure using trichloroacetic acid and sodium deoxycholate to precipitate protein and thus remove soluble substances in the sample that would otherwise interfere in the BCA protein assay. 

Enzyme activities are based on rates of casein hydrolysis under defined conditions. The products of casein hydrolysis, as defined in this protocol, are those peptides soluble in 5% TCA that can be detected by the bicinchoninic acid (BCA) protein assay (unitbi.i). The amount of TCA-soluble peptide generated during the course of the reaction can actually be quantified by any one of several protein/peptide assays. The color yield in these assays is assumed to be proportional to the amount of peptide in solution. The amount of product/peptide in the reaction mixture is often reported as bovine serum albumin (BSA) equivalents—since standard curves based on this protein may be used to calibrate the assay. Thus, activity units can be expressed as the amount of BSA equivalents generated per unit time. 

Additional reagents and equipment for the bicinchoninic acid (BCA) protein assay... 

Remove sufficient supernatant to allow measurement of the soluble peptide by the BCA protein assay (unitbia), using bovine serum albumin (BSA 20 to 120 pg/assay) as a standard. 

Measured as in Basic Protocol 1 with casein substrate. Measured by the BCA protein assay of Smith et al. (1985). 

Bicinchoninic acid (BCA), total protein determination, 77-104 Bilirubin, in BCA protein assay, 96 Binding, water, see Water retention Biological value (BV), protein quality, 128-129, 133 Biuret assay... 

BCA protein assay reagent, effect on, 97 water activity, effect on dew-point measurement, 47-48 generally, 47... 

M NaCl, pH 7.2, containing 10 mM EDTA as the buffer. To obtain efficient separation between the reduced antibody and excess reductant, the sample size applied to the column should be at a ratio of no more than 5% sample volume to the total column volume. Collect 0.5-ml fractions and monitor for protein at 280 nm. To monitor the separation of the second peak (excess Traut s reagent), the BCA protein assay reagent (Pierce) may be used according to the procedure described in the previous section, protocol step 4. 

BCA Protein Assay Kit (Pierce, Rockford, IL) and ELISA plate reader (Dynat-ech MR 5000, Dynatech Labs, Chantilly, VA) for the quantification of cellular protein concentrations. 

Materials Biochemicals were obtained from Sigma Chemical Coip. unless otherwise stated. Sequanal-grade urea and the BCA-protein assay were obtained from Pia ce Chemical Co. All water used was distilled before passage through a MilliQ apparatus. HPLC solvents were obtained from Burdick and Jackson. 

Quantify the concentration of antigen (protein/pDNA) by a micro-BCA protein assay or Hoechst-33258 dye-binding assay. 

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