Lecture 6 & 7

Question 1

What are biochemical techniques used to study proteins?

Answer 1

Step 1: isolating the protein of interest
Step 2: salt precipitation to remove bulk proteins
Step 2: dialysis to remove bulk proteins
Step 3: purifying the protein of interest

Question 2

How does column chromatography work?

Answer 2

a) separation of a protein mixture
b) detection of eluting protein peaks at A 280

Question 3

Size exclusion chromatography (SEC)

Answer 3

separation based on SIZE

also called gel filtration

smaller molecules come out last

Question 4

Ion exchange chromatography

Answer 4

separation based on protein CHARGE as a function of pH

Question 5

Ion exchange chromatography:
Cation:
Anion:
Strength of interaction:
Elution:

Answer 5

Cation: + charged proteins attracted to - charged column material

Anion: - charged proteins attracted to + charged column material

Strength of interaction: determined by # and location of charges

Elution: proteins eluted based on strength of ionic interaction (increase salt concentration of buffer or change pH)

Question 6

In ion exchange chromatography, what happens when the pH is above the pKa?

Answer 6

the UNPROTONATED form predominates (B-)

Question 7

Affinity chromatography

Answer 7

separation based on protein AFFINITY to a ligand

Question 8

What is a ligand?

Answer 8

a molecule that can recognize and bind to the protein

-can be small

Question 9

Why are protein-ligand interactions like?

Answer 9

extremely specific making it a very powerful separation technique

Question 10

Immobilized metal affinity chromatography

Answer 10

separation based on protein having a His6 purification tag

Question 11

Hydrophobic interaction chromatography

Answer 11

separation based on protein surface hydrophobicity

Question 12

Why is Hydrophobic interaction chromatography useful?

Answer 12

proteins bind at high salt concentration and elute at low salt concentration

Question 13

Collecting fractions

Answer 13

computer controlled all in one systems such as the AKTA prime control all aspects of chromatographic separation

Question 14

SDS-PAGE Electrophoresis

Answer 14

smaller molecules come out first

native protein->heat in SDS -> denatured with uniform charge

Question 15

The separation experiment

Answer 15

1) UV absorbance (280 nm) peaks resolved
2) SDS PAGE of fractions
3) correct molecular weight
4) activity assay

Question 16

Isoelectric focusing of native protiens

Answer 16

-polyacrylamide sieving matrix (big molecules move slower)

at the pI, the net charge of a protein is zero it stops moving

acidic proteins have acidic pI’s
basic proteins have basic pI’s

Question 17

2D PAGE Electrophoresis:

IEF followed by SDS PAGE-

Answer 17

-separates on pI then mass
-used in proteome analysis
-can show alternation of gene expression

Question 18

Genome
Transcriptome
Proteome

Answer 18

Genome- all genes (what could happen)
Transcriptome- all mRNA (what might happen)
Proteome- all proteins (what is happening)

Question 19

The proteome is what?

Answer 19

the entire set of proteins expressed by a genome, cell, tissue, or organism under defined set of conditions

Question 20

Zonal centrifugation

Answer 20

low-density and high-density solutions flow into tube with a density gradient

sample layer on top

tube goes into rotor where separation by sediment coefficient occurs

fractions collected through hole in bottom of tube

Question 21

How to determine the shape of a protein:

Answer 21

X-ray diffraction
Nuclear magnetic resonance spectroscopy (NMR)

Question 22

NMR pros and cons

Answer 22

Pros:
no crystals
can do dynamics
can detect protons

Cons:
limited in data
protein size limits
resonance assignments