Topic 2 Purification

Question 1

Two types of purification assessments

Answer 1

Yield Purity

Question 2

Yield

Answer 2

Amount of target protein in final product (mass etc)

Question 3

Purity

Answer 3

Amount of target as a fraction of total protein

Question 4

SDS - PAGE acronym

Answer 4

sodium dodecyl sulfate polyacrylamide gel electrophoresis

Question 5

2 purposes of SDS in SDS PAGE

Answer 5

-Denatures protein -binds to give an even charge to mass ratio

Question 6

SDS structure

Answer 6

Question 7

SDS gives what type of charge ?

Answer 7

Negative

Question 8

2 reducing agents typically used on SDS gels

Answer 8

β-mercapto ethanol

dithiothreitol (DTT)

Question 9

Purpose of reducing agent in SDS gels?

Answer 9

Get rid of disulphide bonds

Question 10

Type of chromatography that separates based on pI

Answer 10

Isoelectric focusing (IEF)

Question 11

Absorbance formula

Answer 11

A = ε c l

Question 12

Aeromatic molecules absorb at approx this wavelength

Answer 12

28 nm

Question 13

Specific wavelengths of aeromatic groups

Answer 13

F - 250 nm

Y - 275 nm

W - 280 nm

Question 14

Typical absorbance relationship with protein concentration

Answer 14

A₂₈₀= 1 when

[protein] = 1 mg ml^-1

Question 15

Bradford assay absorbance

Answer 15

Uses large hydrophobic dye.

Absorbs at 680nm (blue)

Question 16

Enzyme unit

Answer 16

Amount of enzyme that catalyses 1 μM in one minute at 25^oC

(μM / minute)

Question 17

Specific activity

(units / mg)

Answer 17

of enzyme units per mg protein product

Question 18

Exploitable traits for purification (5)

Answer 18

solubility

charge (pI)

size / shape

hydrophobicity

affinity