A pure preparation of a protein is essential, before studying its properties, amino acid composition and amino acid sequencing. The source of a protein is generally tissue or microbial cells. The first step in purifications is to open the cells and to release the proteins into a solution known as a crude extract. Next, differential…
Prepare a homogenate of a biological tissue, liver. Dissect out the liver from an albino rat, dry it, folding between folds of filter paper sheets, and drop it in a beaker kept in ice. Using a pair of scissors, mince it into small pieces. Add about 10 ml of cold distilled water to the mince,…
In biological systems, proteins exist in a variety of forms; some are in free soluble form; some are bound to nucleic acids; and some are bound to lipid-rich membranes. Each type has to be extracted differently.
Isolation of proteins without alteration in their biological function is very important.
This method is about ten times more sensitive than Biuret method. The reagent Folin-Ciocalteu is quite complex and contains phosphomolybdic acid and tungstate. The proteins which contain tyrosine and tryptophan react with these and produce a dark blue colour. A standard protein (BSA) was prepared and used for the assay. This method is based on…
Biuret is the method commonly used. In this method, the CO—NH groups of proteins form a purple complex with copper ions in an alkaline medium. As all proteins contain peptide bonds, this method is fairly specific. Biuret reagent is a mixture of alkaline copper sulphate and sodium potassium tartarate. The cupric ions form a CO—ordination…
During protein purification, the concentration of the protein in the solution should be determined. Colorimetric methods are generally used to determine the protein concentration in the purified sample. The amount of this coloured product is then measured spectrometrically, and the amount of colour related to the amount of protein is present by appropriate collaboration. Various…
The amino acids that are separated from the column should be quantitated by heating them with ninhydrin, which forms purple-coloured compounds. The amount of each amino acid is determined spectrophotometrically by measuring the absorbance of the amino acid derivatives, which is recorded continuously, on a chart recorder or fed directly into a computer for quantitative…
It is an automatic machine to determine the amino acid sequence in a polypeptide chain. The individual amino acids obtained by hydrolysis using Edman’s–Sanger’s method can be separated by cation-exchange chromatography. In this method, amino acids are poured to a column that contains a resin to which a negatively charged group is bound. The amino…
Biuret is a compound formed by heating urea at 180°C.