Electrophoretic Techniques

Question 1

What is the principle of Electrophoresis?

Answer 1

Migration of a charged particle in an electric field

Question 2

What THREE characteristics are related to Electrophoretic Mobility, μep?

Answer 2

1. Net electric charge, q
2. Molecular size / mass, f
3. Molecular shape, f

Question 3

What are the FOUR advantages of Electrophoresis?

Answer 3

1. High Resolution
2. High Sensitivity
3. Multiplicity
4. Simple Set Up

Question 4

What TWO basic electrical equations are important in Electrophoresis?

Answer 4

1. Ohm’s Law: I = E/R
2. Power/Heat Generation: P = EI

Question 5

TRUE OR FALSE:
Only one electrical parameter is held constant in Electrophoresis?

Answer 5

True

Question 6

What do fast Electrophoresis separations require?

Answer 6

High V or I
* create a lot of heat

Question 7

What do high temperatures cause DURING Electrophoresis?

Answer 7

• Denaturation of proteins
• Smile Effect

Question 8

What TWO types of gel are used in VERTICAL Electrophoresis systems?

Answer 8

1. Rods
2. Slabs

Question 9

What type of gel is used in HORIZONTAL Electrophoresis systems?

Answer 9

Agarose

Question 10

What is the role of the gel matrix in EP?

Answer 10

Inhibits convection and diffusion which would impede separation of molecules

Question 11

What is Agarbiose?

Answer 11

Repeating units of agarose
* Galactose
* Anhydrogalactose

Question 12

What concentration is agarose gel used at in EP?

Answer 12

0.7 - 2% w/w

Question 13

What do lower concentrations of agarose gel cause?

Answer 13

Larger pores

Question 14

What does this Ferguson Plot indicate about the protein?

Answer 14

• Same charge densities
• Separation by size

Question 15

What does this Ferguson Plot indicate about the protein?

Answer 15

Protein with higher charge density has smaller size

Question 16

What does this Ferguson Plot indicate about the protein?

Answer 16

Large protein has higher free mobility

Question 17

What does this Ferguson Plot indicate about the protein?

Answer 17

Same size but different mobilities

Question 18

What does SDS do to the protein in EP?

Answer 18

Coats the protein in a NEGATIVE charge

Question 19

What are the THREE parameters of EP separations?

Answer 19

1. SEPARATION - distance between bands
2. RESOLUTION - separation relative to band width
3. SHARPNESS

Question 20

What are the FIVE factors affecting band sharpness in EP?

Answer 20

1. Sample Volume
2. Sample Ion Concentration
3. Diffusion Effects
4. Gel Density
5. Mode

Question 21

What are the THREE characteristics of the Stacking Gel for DISC PAGE?

Answer 21

1. Large pore size
2. Low pH (Tris-HCl)
3. Concentrates into ~20 μm zone

Question 22

What are the THREE characteristics of the Resolving Gel for DISC PAGE?

Answer 22

1. Higher Density
2. High pH (Tris-Glycine)
3. Separation

Question 23

In isoelectrofocusing, what is used to produce gels with pH gradients?

Answer 23

Ampholytes

Question 24

What technique is used for whole cell proteome analysis?

Answer 24

2D Electrophoresis

Question 25

What does the primary antibody do in Western Blotting?

Answer 25

Raised against protein of interest

Question 26

What does the secondary antibody do in Western Blotting?

Answer 26

Recognises primary antibody

Question 27

What label catalyses CL reaction with luminol, enhancer and H2O2?

Answer 27

HRP

Question 28

What is the wavelength of the RED channel in Western Blotting?

Answer 28

680 nm

Question 29

What is the wavelength of the GREEN channel in Western Blotting?

Answer 29

800 nm

Question 30

Which channel are protein markers detected in?

Answer 30

Red channel