SDS-PAGE

Question 0

List 2 reducing agents and their function

Answer 0

Beta-mercaptoethanol (BME)
dithiothreitol (DTT)
Breaks disulfide bonds, which further denatures proteins

Question 1

What are the things to consider during sample prep, specific to the protein structure?

Answer 1

Native vs Denatured structures

Reducing vs non-reducing conditions

Question 2

What do SDS do?

Answer 2

Sodium dodecyl sulfate
Denatures protein and confer uniform -ve charge to protein
Result: all proteins are in their primary structures in rod shapes with uniform -ve charges

Question 3

Summarise 3 methods of denaturing and reducing proteins.

Answer 3

1) . Using SDS
2) . Using reducing agents eg BME and DTT
3) . Heat (60-90deg) before loading

Question 4

What are the functions of the loading buffer?

Answer 4

• Contains glycerol which increases density of solution
• Adds colour to solution (Bromophenol blue tracking dye), which helps with loading process and helps monitor the progress of electrophoretic separation

Question 5

Describe the properties of the PAG

Answer 5

Polyacrylamide Gel
Polymer of Acrylamide monomers
Monomers form long chains in head-to-tail fashion
Bisacrylamide form cross-links between polyacrylamide molecules
Polymerisation initiated by TEMED and APS
Oxygen inhibits polymerization

Question 6

Explain the concept of the Discontinuous Buffer System by explaining the two types of PAGs used

Answer 6

Stacking and Resolving Gel
Stacking: lower pH (6.8), Tris-Cl, Large-pore. Cast over Resolving Gel
Resolving: higher pH (8.8), Tris-Cl, small-pore.
Cl- is the mobile anion in the gels, whereas glycine is the mobile anion in the surrounding tank buffer. Glycine moves into the stacking gel where the pH favours it as a zwittorion (neutral). Cl- in the stacking gel migrates faster than Glycine, and a band is formed between them (region of low conductivity), where the proteins stack). This ensures that they are compact and allow sharp resolution of bands. As the Gly moves into the Resolving gel, the higher pH makes it donate its protons and become anionic, which runs faster, causing proteins to unstack and separate according to their sizes

Question 7

Explain Isoelectric Focusing

Answer 7

Basically introducing a 2nd dimension to separate proteins, ie pH, which can separate different proteins of similar molecular weights. By placing the proteins in a solution of different pH gradients, the protein will move until it is in the region with pH same as its pI eg, if pI is 8, in a pH of 5, protein is +ve charged, and will move towards the cathode (-ve), until it reaches region of pH 8, where is is neutral, therefore will stop migrating

Question 8

Outline the steps of Western blotting.

Answer 8

• Separate proteins by PAGE
• Transfer proteins to nitrocellulose membrane (immobilize)
• Block membrane (milk powder/BSA)
• Add primary Ab
• Add secondary Ab tagged with enzyme (eg HRP)
• Add enzyme substrate, which allows detection of protein band
  (Wash between steps, after Blocking step)

Question 9

Outline the process of immunoprecipitation and co-immunoprecipitation

Answer 9

Using specific Ab and Ab-binding beads in protein mix. The Ab will bind to the bead with the protein of interest bound to the Ab, and it will be pulled-down to the bottom of the tube and may be analysed by PAGE
Co-immunoprecipitation involves pulling down of protein of interest AND associated proteins