Colorimetric Assays

Question 1

In colorimetric assays protein quantitation is achieved by the reaction of

Answer 1

The protein mixture with a specific reagent to produce achromophore

Question 2

Why is colorimetric assays an indirect method

Answer 2

Prior modification of the sample is required priorto measurement, hence the sample cannot be used for further analysis eg activity measurement

Question 3

The most commonly used colorimetric protein assays are

Answer 3

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

Question 4

Biuret and Lowry are cumbersome to perform and have been superseded by

Answer 4

Coomassie and bcaprotein assays

Question 5

Coomassie and Bca are available

Answer 5

Commercially

Question 6

(1) coomassie ( Bradford) protein assay principle: addition of protein to the dye, in an acidic solution serves to

Answer 6

Stabilise the ionic form of the dye by both hydrophobic and ionic interactions

Question 7

(2) The dye in Bradford mainly reacts with

Answer 7

Arginine (r) residues and to a lesser extent with histidine (h), lysine ( k), tyrosine (y)I tryptophan (w) and phenylalanine (f) residues

Question 8

(1b) Bca protein assay: under the alkaline conditions, Cu 2+ forms a complex with

Answer 8

The peptide bonds present in the protein and becomes reduced to Cu+

Question 9

(2 B) bc A acts as a

Answer 9

Cu + capture agent and the entire complex is stable under alkaline conditions

Question 10

In contrast to the uv absorption methods, colorimetric methods require that A–_-___- is prepared

Answer 10

Standard curve 50 that the protein concentrationcan be ascertained