Question

Is there any other way we could obtain the concentration of the protein fractions other than using Bradford assay?

(Hint: Fluorescent proteins)

Answer 1

The calculation of concentration of protein fraction is essential in protein studies. There are different methods have been developed to quantitate both complex mixtures of proteins as well as a single type of protein. Mainly total protein quantitation methods comprise traditional methods like the measurement of UV absorbance at 280 nm, Bicinchoninic acid (BCA) and Bradford assays. There is also some alternative methods like Lowry or novel assays developed by commercial firms. Among the various methods, Individual protein quantitation methods include enzyme-linked immunosorbent assay (ELISA), western blot analysis, and mass spectrometry. In UV analysis excitation at 280 and emission spectra from 330-380 nm is recorded this method is good for both soluble and insoluble protein but your protein should contain tryptophan residues. Dye binding assay (BCA or Bradford or other commercial kits) is another technique which is good for total protein quantification but protein should have positively charged amino acids. The bicinchoninic acid (BCA) assay was invented in 1985 by Paul K. Smith at Pierce Chemical Company, the major distributor of this assay. Both BCA and Lowry assays are based on the conversion of Cu²⁺ to Cu¹⁺ under alkaline conditions. This conversion is called the Biuret reaction and is influenced by several amino acid residues (cysteine, cystine, tyrosine, and tryptophan) and the peptide backbone. The next method Fluorescent dye binding assay is very sensitive. Amino acid analysis is method which is good but laborious.

Lowry protein assay is for proteins in the range: 10-1000 ug/ml. Lowry assay, proposed by Oliver H. Lowry in 1951, is based on two chemical reactions. The first reaction is the reduction of copper ions under alkaline conditions, which forms a complex with peptide bonds. The second is the reduction of Folin-Ciocalteu reagent by the copper-peptide bond complex, which subsequently causes a color change of the solution into blue with an absorption in the range of 650 to 750 nm detectable with a spectrophotometer. The amount of protein in the sample can be estimated using a standard curve of a selected standard protein solution such as in BSA.

The 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) assay is technique in which range of proteins is limited to 100 -1500 μg /ml. 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) is a sensitive fluorogenic reagent used for the detection of amines in proteins. Because it can detect only the accessible amines in the protein assayed, this method has the same limitations of spectrophotometric methods for which the outcome depends on the number of certain amino acids in the protein. However, CBQCA is very sensitive and can detect from 10 to 150 ug of protein in a 100 ul volume. Also on the positive side, the CBQCA reagent functions well in the presence of substances, such as lipids, known to interfere in many other protein determination methods, or when used to quantify surface-attached or encapsulated peptides or proteins.