SDS Gel Electrophoresis

Question 1

What are protein?

Answer 1

macromolecules that play crucial role in virtually all biological processes

Question 2

5 biological processes that involve protein

Answer 2

1. enzyme catalysis
2. transport and storage
3. mechanical support
4. immune protection
5. control of growth and differentiation

Question 3

Basic protein structure

Answer 3

• primary structure: amino acid sequence
• secondary structure: alpha-helix, beta-sheet, turns and random coils
• tertiary structure: secondary structure motif combinations
• quaternary structure: arrangement of multiple folded subunits to form final structure

Question 4

Basis of electrophoresis

Answer 4

separate proteins according to their molecular weight

Question 5

SDS-PAGE means:

Answer 5

sodium dodecyl sulphate poly-acrylamide gel electrophoresis

Question 6

What kind of detergent is SDS and what charge does it have?

Answer 6

anioinc

-ve

Question 7

What effect does SDS have on protein? (4)

Answer 7

• makes protein linear (rod shaped)
• unfolds proteins so they only have a primary structure
• masks any net charge of protein by forming large anionic complexes
• disrupts H-bonds, blocks hydrophobic interactions

Question 8

Why are reducing agents added to things?

Answer 8

• break disulphide bonds

- detach subunits to ensure a separation based on molecular weight

Question 9

Common reducing agents (2)

Answer 9

• beta-mercaptoethanol

- dithiothreitol

Question 10

Polyacrylamide

Answer 10

• polymer of acrylamide monomers + bis-acrylamide
• once polymerised a gel forms
• gel made of labyrinth of different sized tunnels through a meshwork of fibres

Question 11

How do differently sized protein move through polyacrylamide gel?

Answer 11

• smaller ones more faster
• bigger ones move slower
• they’re separated according to molecular weight

Question 12

What’s applied to gel for proteins to move through it?

Answer 12

voltage

Question 13

Discontinuous gel electrophoresis

Answer 13

two different gels (one on top of the other) with different pH and acrylamide concentrations

1. stacking gel
2. resolving gel

Question 14

Discontinuous gel electrophoresis stacking gel composition

Answer 14

• pH 6.8

- 4% acrylamide

Question 15

Discontinuous gel electrophoresis resolving gel composition

Answer 15

• pH 8.8

- varying % of acrylamide, normally ~ 10-15%

Question 16

When do proteins separate during discontinuous gel electrophoresis?

Answer 16

when they enter the resolving gel

Question 17

Gradient gel electrophoresis

Answer 17

Proteins migrate through an increasing % of acrylamide and gel becomes increasingly restrictive for proteins to migrate

Question 18

Staining gels

Answer 18

to visualise proteins they first need to be immobilised in weak acid and then stained with Coomassie blue and then destained to remove excess dye

Question 19

Molecular weight determination

Answer 19

Run a gel lane with protein of known molecular weight and draw a standard curve of LogMW and Rf