BCA assay

Furthermore, every protein determination is sensitive to detergents or certain ions. Hence, when presenting a concentration value it is good practice to also mention the assay that was used as well as the benchmark protein. The methods of choice are the Bradford assay and the BCA (bicinchoninine acid) assay. Anyone working with membrane proteins and detergents should use the BCA assay. Otherwise, the choice between the BCA and the Bradford assays seems to be a question of taste. 

Proteins form a complex with Cu ions in alkaline solution (Biuret reaction). The Cu ions of the complex are presumably reduced to Cu ions, which form a violet color complex with BCA. 

Advantages The test is quick (10 minutes at 65° C), sensitive (detection threshold 0.5 pg of protein), and resistant to detergents such as TRlTON-X-100. The reaction takes place in an 

Problems It is disrupted by high concentrations of complex-forming reagents (e.g., EDTA) and by ammonium sulfate N-acteyl-glucosamine, glycine, reducing materials such as glucose, DTT, or Sorbitol and a host of pharmaceuticals such as chlorpromazine, penicillin, and vitamin C (Marshall and Williams 1991). 

Marshall, T., and Williams, K. (1991). Drug Interference in the Bradford and 2,2 bicinchoninic Acid Protein 

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Calculate the protein concentration in the final preparation using its absorbance at 280nm or a colorimetric method, such as the Coomassie assay. (Note The presence of hydrazine or hydrazide groups on the protein will interfere with the BCA assay for total protein concentration.)... 

Measure the absorbance of the biotinylated protein solution at 354 nm. Use the molar extinction coefficient for the chromogenic group (e = 29,000 M-1cm-1) to determine the concentration of biotin present. To determine the molar ratio of biotin-to-protein, divide the molar concentration of biotin by the molar concentration of protein present (which may be determined by using the Coomassie assay or the BCA assay methods). 

The BCA assay has a working range of 20 to 2000 Lig/ml. If the target protein is in a dilute aqueous formulation, the concentration can be determined with the micro BCA assay. The BCA protocol is modified by increasing the BCA concentration, incubation time, and incubation temperature. These modifications permit the detection of BSA at 0.5 Lig/ml. The major disadvantage of these modifications is that the presence of interfering substances decreases the signal-to-noise ratio and thus the sensitivity of the assay. 

The BCA assay for immobUized protein is more reliable than calculations from UV measurements of coupling supernatant. 

Increasing the incubation temperature to 60°C lowers the minimum detection level to 5 fig/ml and narrows the working range of the assay to a maximum of 250 fig/ml. This is known as the enhanced BCA assay. 

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. 

Akins, R.E. and Tuan, R.S. 1992. Measurement of protein in 20 seconds using a microwave BCA assay. Biotechniques 12 496-499. 

The reducing sugar-based assays are predicated on the correct measurement of enzymegenerated product. A typical calibration curve for the BCA assay, using galacturonic acid as the calibration standard, is presented in Figure Cl.2.5. Note that the assay is linear for the... 

Bovine gamma globulin (BGG), total protein determination BCA assay, 84 (fig.) biuret assay, 88 (fig.), 98 Coomassie plus assay, 90 Bovine serum albumin (BSA), total protein determination BCA assay, 84 (fig.) biuret assay, 88 (fig.), 98 Coomassie plus assay, 90 protein standards, 78... 

Detection Limit, Linear Range and Working Range for DNS, DNSsg, Nelson-Somogyi (Nelson 10 mL), Modified Nelson-Somogyi (Nelson 5 mL), PAHBAH," and BCA Assays... 

Cellodextrin Reponses for DNS, Nelson-Somogyi (Nelson), and BCA Assays... 

The combined data provide a ready means by which to compare and select appropriate assays for application in cellulase-catalyzed cellulose saccharification experiments. Products in such experiments are expected to include glucose cellobiose and, potentially, some cellooligosaccharides. Optimum reducing sugar assays would have equivalent molar color yields for these soluble products. As shown in Table 3, this optimum situation only applies to the two copper-based assays (Nelson, BCA). Because of their importance with respect to the analysis of insoluble cellulose (discussed next), calibration curves reflecting the molar color yields for the DNS and BCA assays are presented in Fig. 1. 

Fig. 1. Absorbance response of equimolar amounts of soluble cellulose saccharification products glucose (G), cellobiose (G2), and cellotriose (G3) by (A) DNS assay and (B) BCA assay.

Fig. 2. Reducing groups per amount of cellulose determined by BCA assay for MCC ( ), AMCC ( ), PSC (A), BMCC, (x), and A BMCC, ( ). Values represent the mean of triplicate determinations error bars represent the SE. When not shown, the error bars fall within the symbols.

Fig. 3. Reducing groups per amount of filter paper determined by BCA assay. The statistical parameters are as in Fig. 2.

Fig. 6. Time courses of reduction of celluloses by NaBH4 at pH 8.0,22°C. Unreacted reducing ends were determined by BCA assay. The celluloses are as in Table 4. FP, filter paper.

Total protein is measured using the bicinchoninic acid method (BCA assay Pierce Co., Rockford, IL). 

Bicinchoninic Acid (BCA) Assay. The bicin-choninic acid assay for proteins is based on the same reactions as the Folin-Ciocalteau assay. Proteins are again reacted with alkaline cupric ions to form the biuret complex, and these ions are reduced to cuprous ions by the aromatic amino acids in the proteins. In this case, however, the Cu1+ ions form a complex with bicinchoninic acid (Fig. II-6), which has an intense absorbance maximum at 562 nm. This assay shows the same variation from protein to protein as the Folin-Ciocalteau assay, but is more convenient experimentally and can be made somewhat more sensitive. 

Walker, J.M. (1994b) The Bicinchonic (BCA) Assay for Protein Quantitation, Methods Md. Biol. 

Prepare the assay reagent according to the instructions in the Pierce micro BCA assay kit. 

The protein content of the folate conjugate may be determined by a standard protein assay, e.g., using the bicinchoninic acid (BCA) assay (available from Pierce Chemical Co., Rockford, IL) (8). The number of folates attached to the protein can be determined by measuring ultraviolet absorption of the conjugate solution at 363 nm. The extinction coefficient for folate at that wavelength in PBS is 6197 (8). The number of folates per protein can, therefore, be calculated by the following equation ... 

The filtered XlnD fermentation broth was initially concentrated into 20 mM Bis-Tris buffer at pH 6.8 in a 300 ml stirred ultrafiltration cell from Amicon (Beverly, MA) with a 30,000 Da molecular weight cut-off membrane. The protein was then bound to a Source Q anion exchange column (96 ml) and eluted over a 0 to 1 M NaCl gradient into 20 mM Bis-Tris at pH 6.8. The eluted XlnD peak was then reconcentrated and rerun on the Source Q column. From this run, the XlnD fractions were then pooled and buffer-exchanged into 20 mM acetate buffer at pH 5.0 with 100 mM NaCl on a Superdex 200 size exclusion column. The purity of the purified fiactions was assessed by SDS-PAGE, and the protein content was assessed by measuring the absorbance at 280 nm and by bicinchoninic acid (BCA) assay with bovine serum albumin (BSA) run as a standard. 

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