L11

Question 1

Three stages in protein purification

Answer 1

1. sample homogenization - physical disruption of cells or tissues
2. differential centrifugation to isoalte different cellular components
3. separation of proteins

Question 2

What is the basis for separating proteins?

Answer 2

• solubility
• size
• charge
• hydrophobicity
• affinity for ligands

Question 3

Does high or low temp help with grinding and separating tissues

Answer 3

low temp

Question 4

what is the formula for acceleration?

Answer 4

a = w^2 x r
(w = angular velocity (radians/sec)
(r = radius in metres)

Question 5

What is crude separation?

Answer 5

proteins being precipitates by adding ‘competing solutes’ like ammonium sulphate, acetone, or polyethlene glycol

Question 6

Ideally, competing solutes should be:

Answer 6

• very soluble in water
• relatively non-denaturing to proteins
• easy to remove from the proteins afterwards
• not too viscous or dense
• cheap and pure

Question 7

What are the stationary and mobile phases of chromatogrphy

Answer 7

stationary phase : pigments loaded on bottom of filter paper
mobile phases : organic solvent diffused from bottom up

Question 8

What are the basic steps of chromatography?

Answer 8

1. sample is added to stationary phase
2. flow of mobile phase takes the sample through the column
3. different substances separate and appear at the bottom of the column after different volumes have been added (elution volumes)

Question 9

How does size exclusion chromatograph (SEC) work?

Answer 9

there are carbohydrate polymer beads and molecules are added - small molecules enter the aqueous spaces within beads whereas larger molecules cannot enter the beads

Question 10

Separation on the basis of charge - ion exchange chromatography

Answer 10

positively charged protein binds to negatively charged bead and negatively charged protein flows through

Question 11

affinity chromatography

Answer 11

immobilized molecule with affinity for the protein is used to trap protein of interest in the column

Question 12

define eluting

Answer 12

elution is the process of extracting one material from another by washing with a solvent

Question 13

What is immunoaffinity chromatography?

Answer 13

antibody against protein of interest

Question 14

Immobilized ligan chromatogrphay

Answer 14

substate analogue or inhibitor binds enzyme or protein (i.e. heparin and heparin-binding proteins)

Question 15

lectin-based affinity chromatography

Answer 15

lectins bind glocosylated proteins - elution is via addition of sugars (i.e. N-acetyl glycosamine)

Question 16

Immobilized metal affinity chromatography

Answer 16

metal ions (i.e. Ni2+) bind engineered recombinant proteins containing a poly-histidine tag at the N- or C- terminus

Question 17

Where do sequence numbers start from ?

Answer 17

The N-terminus