Electropheresis

Question 1

What is isoelectric point?

Answer 1

• Protein has no net charge at this pH

Zwitterion

Question 2

What does R group determine?

Answer 2

R group determines whether amino acid is neutral, acidic or basic

Question 3

What is electrophoresis and what does it depend on?

Answer 3

• Migration of charged particles in electric field
○ Based on size, shape or charge
Current and resistance

Question 4

Why is electrophoresis useful?

Answer 4

Useful for separating and purifying macromolecule

Question 5

Apparatus involved in electrophoresis?

Answer 5

○ Horizontal-Usually for agarose gel

Vertical-for polyacrylamide gel

Question 6

What to ensure in electrophoeresis

Answer 6

○ Ensure:
§ Uniform electric field across gel
§ Cooling to prevent thermal artefact
Access to gel loading and monitoring

Question 7

Gel electropheresis

Answer 7

○ Gel electrophoresis:
§ Cast in shape of thin slab with wells
§ Immersed within buffer:
□ Maintain relatively constant pH
□ pH of solution and nature of R groups have important effect on migration of proteins
Proteins separated within gel with series of pores

Question 8

Types of electrophoretic gel(Agarose)

Answer 8

○ Polysaccharide extract from sea weed
○ Prepared by dissolving powdered agarose in buffer
○ Heat and pour into casting tray
○ Undergoes polymerisation when cooled
○ Pores relatively larger, so relatively low resolving power
○ Used to separate large proteins >200kDa
Used at concentrations of 0.5-2%

Question 9

Types of electrophoretic gel(Polyacrylamide)

Answer 9

○ Formed from synthetic small molecule acrylamide
○ Polymerises into long chains in presence of catalyst and initiator(APS and TEMED)
○ Polyacrylamide gel have smaller pores than agarose

Pore size also determine by polyacrylamide concentration

Question 10

What are buffers?

Answer 10

Supply current carrying ions in electrophoretic cell

Question 11

What do buffers maintain?

Answer 11

Maintain pH

Question 12

What do buffers provide a medium for?

Answer 12

Provides medium for heat dissipation

Question 13

Classification of buffers>

Answer 13

○ Either classified as continuous or discontinuous
○ Continuous:
§ Use same buffer in gel, sample and tank
○ Discontinuous:
§ Non-restrictive large-pore gel
§ Resolving gel-greater resolution
§ Have different buffers for stacking gel, resolving gel and tank buffer