Question

4. Bradford assay. In your prac you used the Bradford assay to determine the protein concentration (1.5 marks)

a) Explain at a molecular level how the color development of the assay works. Answer must be less than 100 words. (0.5 marks)

b) Explain why you can use Bovine Serum Albumin as standard protein to determine the concentration of milk proteins. Answer must be less than 50 words. (0.5 marks)

c) Why is it important to use diluted milk fraction samples when performing the Bradford analysis? Answer must be less than 50 words. (0.5 marks)

Answer 1

a) At molecular level how the colour development of assay works - So, the Bradford assay which is colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Briliant blue G-250.The Coomassie Brilliant blue G-250 dye exists in three forms - anionic (blue), neutral ( green ) and cationic (red).Under acidic conditions the red form of the dye is converted into its blue form, binding to the protein being assayed.If there is no protein to bind then the solution will remain brown.The dye forms a strong noncovalent complex with the proteins carboxyl group by Vander Waals force and amino group through electrostatic interactions.

b)BSA is used because of its ability to increase signal in assays its lack of effect in many biochemical reactions, and low cost since large quantities of it can be readily purified from bovine blood , a byproduct of the cattle industry.It is also used to determine the quantity of other proteins.

c)It is important to use diluted milk because the Bradford assay is calibrated by using a pure protein of known concentration called the standard protein.The Bradford assay is as sensitive to BSA as IgG as to BSA. So it is best to use the protein for the standard which we wish to estimate in the unknown.And the diluted milk is used for the correction factor of the unknown protein.