Colorimetric Assays Flashcards
In colorimetric assays protein quantitation is achieved by the reaction of
The protein mixture with a specific reagent to produce achromophore
Why is colorimetric assays an indirect method
Prior modification of the sample is required priorto measurement, hence the sample cannot be used for further analysis eg activity measurement
The most commonly used colorimetric protein assays are
Coomassie blue (Bradford) protein assay A 590 nm
Bichinchoninic acid (bca) protein assay A 562 nm
Biuret protein assay A 540 nm
Lowry Protein assay A 600 - 750 nm
Biuret and Lowry are cumbersome to perform and have been superseded by
Coomassie and bcaprotein assays
Coomassie and Bca are available
Commercially
(1) coomassie ( Bradford) protein assay principle: addition of protein to the dye, in an acidic solution serves to
Stabilise the ionic form of the dye by both hydrophobic and ionic interactions
(2) The dye in Bradford mainly reacts with
Arginine (r) residues and to a lesser extent with histidine (h), lysine ( k), tyrosine (y)I tryptophan (w) and phenylalanine (f) residues
(1b) Bca protein assay: under the alkaline conditions, Cu 2+ forms a complex with
The peptide bonds present in the protein and becomes reduced to Cu+
(2 B) bc A acts as a
Cu + capture agent and the entire complex is stable under alkaline conditions
In contrast to the uv absorption methods, colorimetric methods require that A–_-___- is prepared
Standard curve 50 that the protein concentrationcan be ascertained
