Biuret protein assay

Preparation of Tubes for the Biuret Protein Assay on Mitochondrial Fractions1... 

Table B1.1.3 Troubleshooting Guide for Biuret Protein Assay...

Review methods for protein assay including absorbance and colorimetric (Lowry, Biuret, Bradford, BCA). 

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. 

The biuret total protein reagent is considerably less sensitive to total protein than the other three protein assay reagents discussed in this unit. This limits the applications in which the Biuret reagent can be used. Since the primary use of the Biuret reagent has been for serum total protein in the clinical laboratory, there is little published information about its compatibility with substances and reagents common to nonclinical samples. 

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... 

In the Biuret reaction, a purple colour develops when the protein is treated with alkaline copper sulphate. This reaction is dependent on peptide bonds and not on the side chains of individual amino-acids present. In the Folin-Ciocalteu reaction, the protein is treated with tungstate and molybdate under alkaline conditions and the formation of a complex such phenylalanine and tyrosine gives rise to a blue colour. Lowry developed one of the most widely used protein assays in which a combination of the above reactions is involved07, l8). 

Figure 2-14. Absorbances observed with increasing amounts of protein assayed by means of biuret reagent.

The abundance and ease of purification made bovine serum albumin (BSA) an early standard in protein chemistry, and BSA is widely used as protein standard in biuret, Lowry, and Bradford assays as well as a molecular weight... 

Estimate the protein concentration using the biuret assay. If necessary, dilute to 1 mg protein/mL. 

Basic Protocol 3 The Biuret Assay for Determining Total Protein Bl.l. 10... 

Alternate Protocol 5 Microtiter Plate Biuret Assay for Total Protein B1.1.13... 

MICROTITER PLATE BIURET ASSAY FOR TOTAL PROTEIN... 

Beyer, R.E. 1983. A rapid biuret assay for protein of whole fatty tissues. Anal. Biochem. 129 483-485. 

Watters, C. 1978. A one-step biuret assay for protein in the presence of detergent. Anal. Biochem. 88 695-698. 

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... 

Protein Determinations The biuret assay (23) was employed for determining protein in tissue samples. 

The Folin-Ciocalteau Assay of Protein Concentration. The Folin-Ciocalteau assay is one of the most sensitive and most commonly used assays to determine protein concentration (sensitive to about 10 /rg/m I protein). This procedure employs two color-forming reactions to assay protein concentration photometrically. In the first reaction (a biuret reaction), compounds with two or more peptide bonds form a dark blue-purple color in the presence of alkaline copper salts. In the second reaction, tryptophan and tyrosine side chains react with the Folin solution to produce cuprous ions. This reaction is most efficient under basic condi-... 

Biuret Test. Compounds containing two or more peptide bonds (e.g., proteins) take on a characteristic purple color when treated with dilute copper sulfate in alkaline solution. The name of the test comes from the compound biuret, which gives a typically positive reaction. The color is apparently caused by the coordination complex of the copper atom and four nitrogen atoms, two from each of two peptide chains (Fig. II-5). The biuret test is fairly reproducible for any protein, but it requires relatively large amounts of protein (1 to 20 mg) for color formation. Because of its low sensitivity, the biuret assay is no longer widely used. 

Folin-Ciocalteu (Lowry) Assay. The quantitative Folin-Ciocalteu assay (also often called the Lowry assay ) can be applied to dried material as well as to solutions. In addition, the method is sensitive samples containing as little as 5 /Ug of protein can be analyzed readily. The color formed by the Folin-Ciocalteu reagent is thought to be caused by the reaction of protein with the alkaline copper in the reagent (as in the biuret test) and the reduction... 

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. 

---