Bicinchoninic acid assays

This unit describes four of the most commonly used total protein assay methods. Three of the four are copper-based assays to quantitate total protein the Lowry method (see Basic Protocol 1 and Alternate Protocols 1 and 2), the bicinchoninic acid assay (BCA see Basic Protocol 2 and Alternate Protocols 3 and 4), and the biuret method (see Basic Protocol 3 and Alternate Protocol 5). The fourth is the Coomassie dye binding or Bradford assay (see Basic Protocol 4 and Alternate Protocols 6 and 7), which is included as a simple and sensitive assay, although it sometimes gives a variable response depending on how well or how poorly the protein binds the dye in acidic pH. A protein assay method should be chosen based on the sensitivity and accuracy of method as well as the condition of the sample to be analyzed. 

Colorimetric assays are appropriate methods to investigate protein interactions with cationic polysaccharide materials. However, these assays cannot differentiate between different types of interactions, i.e. adsorption and absorption of proteins. Among these, the bicinchoninic acid assay (BCA) is widely used since it allows an easy determination of the total amount of adsorbed proteins on bulk materials." The assay is based on the reduction of Cu to Cu in a reductive environment in the presence of proteins. The BCA chelates Cu, forming a colored complex whose absorption band at 562 nm can be related to the amount of adsorbed proteins on the materials. Using this method, it has been demonstrated that the incorporation of inorganic materials such as hydroxyapatite (HAP) or nano-HAP into chitosan-based materials leads to the formation of protein-rich layers at the... 

Alkaline phosphatase (ALP) activity ALP activity of the supematarrt was meastrred using an ALP B-Test WAKO kit (Wako, Japan) based on the Bessey-Lowry method. ALP activity was normalized by samples total protein production measured with a Bicinchoninic Acid Assay (BCA, BCA Protein Assay KiL Pierce Biotechnology, U.S. A.). 

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. 

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 ... 

Wiechelman. K.. Braun, R., and Fitzpatrick, J. 1988. Investigation of the bicinchoninic acid protein assay Identification of the groups responsible for color formation. Anal. Biochem. 175 231-237. 

Figure C1.2.5 Calibration curve for the 2,2 -bicinchoninic acid reducing sugar-assay with galac-turonic acid as the calibration standard and A560 as the analytical signal.

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... 

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... 

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. 

Figure II-6 Chemistry of the bicinchoninic acid protein assay.

Monitoring of solubility and dissolution Gravimetric analysis, a protein assay method (involving color reaction with bicinchoninic acid) and UV absorbance measurements at 214 nm Chemical analysis using TNBS reagent Fluorescence spectrophotometry... 

The three most common assays for total protein2 are the Lowry (enhanced copper), Smith (bicinchoninic acid, BCA), and Bradford (Coomassie Blue) methods. All are colorimetric methods, and are based on the generation of absorbing species in proportion to the quantity of protein present in the sample. The ninhydrin assay is a recently reported promising method. 

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. 

Wiechelmann, K., et al. (1988). Investigation of the Bicinchoninic Acid Protein Assay Identification of the... 

BSA was used as model protein drug to determine the release behavior of protein or peptide from protein loaded composite hydrogel in vitro. The detail procedure was similar to the study of VB12 release, but the initial drug loading amount were 4mg. The amount of BSA present in the supernatant was determined by bicinchoninic acid (BCA) assay and BCA Protein Assay Kit (PIERCE, USA) was used. The SDS-polyacrylamide gel electrophoretic (PAGE) analysis was used to assay the stability of BSA during the release period. 

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