Colorimetric Assays and Standard Curves: The Bradford Assay

Question 1

What is a protein assay ?

Answer 1

Quantitation of protein in a sample

Question 2

When would a protein assay be used ?

Answer 2

1. During a purification procedure
2. At the end of a purification procedure
3. During enzyme kinetic studies
4. During binding studies
5. Comparative studies

Question 3

What can a prtoein assay tell us ?

Answer 3

1. Yield
2. Fold purification
3. Specific activity

Question 4

What is the equation for specific activity ?

Answer 4

Activity of an enzyme/Protein concentration

Question 5

What are the units of specific activity ?

Answer 5

Units per mg

Question 6

If the enzyme is pure, what is the specific activity ?

Answer 6

Characteristic of that enzyme

Question 7

What does low specific activity suggest ?

Answer 7

1. Lots of contaminating protein
2. Lots of inactive enzyme

Question 8

What are some considerations when selecting a protein assay ?

Answer 8

1. Is the assay fit for purpose
2. Is the assay reliable
3. Do you have the necessary instrumentation
4. Is there a cost implication
5. Is it fast
6. Do reagents used in the purification interfere with the assay
7. Has the method the required sensitivity
8. Is the method destructive

Question 9

What does protein absorb light maximally at ?

Answer 9

280nm

Question 10

Why does protein absorb light maximally at 280nm ?

Answer 10

Due to the presence of tyrosine and tryptophan

Question 11

What and why does phenylalanine absorb at ?

Answer 11

260nm due to aromatic ring

Question 12

When can you use the beer lambert law to accurately determine the protein concentration ?

Answer 12

If you have a pure protein and you know the sequence of the protein

Question 13

What is cysteine ?

Answer 13

The product of the cross linking of two cyesteine residues

Question 14

What are colorimetric asssays based on ?

Answer 14

The use of a reference/standard protein

Question 15

What considerations apply to colorimetric assays only ?

Answer 15

1. Can you assume that the reference protein and your protein behave in identical ways ?
2. Are you working within the linear range of the standard curve ?
3. By its nature, the method is destructive, you will sacrifice protein, how much can you spare ?

Question 16

What does the Bradford Assay involve ?

Answer 16

The addition of acidic dye, coomassie brilliant blue G-250 to the protein solutions

Question 17

What does the coomassie dye bind to and what is the result ?

Answer 17

The dye binds to basic and aromatic amino acids resulting in a shift of the absorbance maximum from 465nm (brown) to 595nm (blue)

Question 18

What is chromatography ?

Answer 18

A method for separating the components of a mixture on the basis of differences in physical of chemical characteristics

Question 19

What type of chromatography for molecular mass ?

Answer 19

Gel filtration

Question 20

What type of chromatography for charge ?

Answer 20

Ion exchange

Question 21

What type of chromatogrpahy for specific bonding ?

Answer 21

Affinity chromatography

Question 22

What types of molecules come out first of gel filtration chromatography ?

Answer 22

Large molecules

Question 23

What is pI of protein ?

Answer 23

The point at which there is no net charge on the protein

Question 24

What is a charge above pI ?

Answer 24

Positive

Question 25

What is charge below pI ?

Answer 25

Negative