Bradford Assay (Experiment 4.1)

Question 1

How can the concentration of a protein be determined?

Answer 1

Concentration of a protein can be estimated by measuring the OD at 280 nm due to the presence of Trp, Tyr, and Phe.

Question 2

How can the concentration of a protein be determined?

Answer 2

Concentration of a protein can be estimated by measuring the OD at 280 nm due to the presence of Trp, Tyr, and Phe.

Question 3

What are some of the reasons that the Bradford Method is not very accurate?

Answer 3

1. composition of protein varies greatly.
2. proteins with none of these amino acids (Trp, Tyr, and Phe) do not have maximum absorption at 280 nm.
3. Nucleic acid contamination can also interfere.

Question 4

Which dye does the Bradford method use?

Answer 4

Coomassie Brilliant Blue that is prepared as a stock solution in phosphoric acid.

Question 5

Why the Coomassie Brilliant Blue Dye?

Answer 5

The Coomassie dye forms non-covalent complexes with proteins through electrostatic and Van der Waals interactions with the protein causing a red shift in the absorbance of the dye from 465 nm (red) to 595 nm (blue).

Question 6

What is the purpose of protein quantification?

Answer 6

To determine the progress of a protein from a mixture of other proteins.

Question 7

What is the purpose of protein purification?

Answer 7

is to increase the specific activity of the enzyme. If a purification is proceeding properly and the enzyme becomes more pure the specific activity will increase.

Question 8

How do we determine concentration of protein from BSA Standard curve?

Answer 8

conc. unknown (ug/ml) = A595 nm of unknown/m (slope of standard curve).