SFP4: Protein Gel Electrophoresis And Western Blotting

Question 1

Name two methods for assessing protein purity

Answer 1

Column chromatography

Electrophoresis (SDS PAGE)

Question 2

How does column chromatography prove protein purity?

Answer 2

They provide analytical information on the proteins
E.g. Gel filtration/size-exclusion
Number of peaks = number of proteins in the mixture

A280 is proportional to amount of protein (conc.) so can assess relative purity (area under curve)
See lecture if not understood

Question 3

How does gel electrophoresis assess protein purity?

Answer 3

Separates differences in DENATURED weight

Question 4

What is the process of SDS PAGE

Answer 4

Protein samples are denatured
Treated with SDS
Binds to and gives proteins overall negative charge
Apply voltage through polyacrylamide gel
Smaller proteins move faster

Question 5

Is the anode or cathode at the bottom of the gel?

Answer 5

Anode as it attracts the negatively charged proteins

Question 6

What is used to denature proteins for SDS PAGE?

Answer 6

1. Urea- disrupts non covalent interactions in proteins
2. Beta-mercaptoethanol - used to reduce disulphide bonds
3. SDS (sodium dodecyl sulphate) -detergent
   Negatively charged, native protein unfolds, constant charge:mass ratio

Question 7

How many different proteins for resolution does 2D gel electrophoresis allow up to?

Answer 7

Up to 4000 different proteins

Question 8

What is first dimension isoelectric focussing?

Answer 8

Separation of proteins depending on their isoelectric points (pI)

Question 9

What is the isoelectric point of a protein?

Answer 9

The pH at which a particular protein has no net overall charge (depends on -R group)

Question 10

Is a pI of 4 acidic or basic?

Answer 10

Acidic

Question 11

What is 2nd dimension of gel electrophoresis?

Answer 11

SDS Page (denature proteins then separate by weight)

Question 12

What does the intensity of a spot on the gel relate to?

Answer 12

The abundance of the protein

Question 13

What is proteomics?

Answer 13

Proteomics is analysis of protein expression (proteomes)

Question 14

Name one application of expression proteomics?

Answer 14

Can be used to identify disease bio markers

May be useful in diagnosis, can inform about disease mechanism