Exp 2.4: Chromatography and Assay

Question 1

used to separate and identify components or solutes in a mixture

Answer 1

chromatography

Question 2

principle of chromatography

Answer 2

based on affinity

Question 3

a solid-liquid technique in which the two phases are a solid and a liquid

Answer 3

thin layer chromatography (TLC)

Question 4

Identify the phases in TLC:
solid
liquid

Answer 4

solid: stationary phase
liquid: mobile phase

Question 5

Identify the substances used:
stationary phase
mobile phase

Answer 5

stationary phase: silica gel
mobile phase: ButOH:HOAc:H2O (4:1:5)

Question 6

used to determine the amino acid concentration of a given protein solution

Answer 6

chromatography

Question 7

used in chromatography visualization

Answer 7

ninhydrin

Question 8

retention factor (rf) formula

Answer 8

rf = distance travelled by the amino acid (cm) / distance travelled by the solvent (cm)

Question 9

stages in thin layer chomatography

Answer 9

1. sample/ std. application
2. development
3. visualization
4. evaluation
5. documentation

Question 10

a simple colorimetric and one of the most commonly used assays for total protein concentration

Answer 10

Bradford assay

Question 11

basis of Bradford assay

Answer 11

proportional binding of Coomassie dye to proteins

Question 12

principle in Bradford assay

Answer 12

In an acidic medium, proteins bind to coomassie dye due to hydrophobic and electrostatic interactions

Question 13

λ max of dye

Answer 13

465 to 595 nm

Question 14

To what type of amino acids does Coomassie Brilliant Blue G-250 (dye) react?

Answer 14

basic (primarily Arg) and aromatic amino acids

Question 15

How much protein does Bradford assay measure?

Answer 15

10-100 mg protein

Question 16

standard used in Bradford assay

Answer 16

bovine serum albumin (BSA)

Question 17

dye dissolved in ethanol and phosphoric acid

Answer 17

Bradford reagent