lecture 10 - electrophoresis

Question 1

How does gel electrophoresis work?

Answer 1

• charged proteins separate in an electric field

Question 2

What does the protein of charge q feel in the electric field E

Answer 2

• Feels an attractive force towards the electrode

- FE= Fq

Question 3

What is the frictional force F opposed to and proportional to?

Answer 3

• protein size
• velocity
• viscosity

Question 4

what is stokes law ?

Answer 4

Velocity = 6phr

Question 5

What does Stokes law depend on?

Answer 5

• the size of the protein

Question 6

What is the apparatus needed for the electrophoresis ?

Answer 6

• pair of platinum electrodes on either side of the buffer reservoir , into which is placed a solid supporting medium which may be inert

Question 7

why do we have to use a buffer?

Answer 7

to keep the pH the same

Question 8

What happens when the pH = pI?

Answer 8

the protein will not move , the charges are the same

Question 9

Why must the solid supporting matrix of the apparatus be thin?

Answer 9

• minimize convection and denaturation

Question 10

Why two types of solid supporting matrixes can you have?

Answer 10

• horizontal or vertical

Question 11

What are 2 factors that the matrix must be?

Answer 11

• inert

- porous

Question 12

Why must the matrix be inert?

Answer 12

• the protein does not interact with it

Question 13

Why must the matrix be porous?

Answer 13

allow separation for charge and size

Question 14

What is the most common gel matrix used?

Answer 14

polyacrylamide, (agarose, if protein is very large)

Question 15

What are the 3 ways you can detect protein on a gel matrix?

Answer 15

• Coomassie Brilliant Blue
• Silver Stain
• Specific detection

Question 16

what are the chemicals used for Coomassie Brilliant blue and what are the measurement it can detect?

Answer 16

• In MeOH/CH3COOH to denature & fix
• Destain in same without dye
• Detects 1mg-100ng

Question 17

What volume can silver stain detect?

Answer 17

1ng

Question 18

What does PAGE stand for?

Answer 18

Polyacrylamide Gel Electrophoresis

Question 19

How is the PAGE gel formed?

Answer 19

Acrylamide polymer
with N,N’-methylene bisacrylamide crosslinks,
Catalysed by SO4-•

Question 20

How big can the pore size be in the PAGE gel?

Answer 20

• depends on the concentration of the acrylamide

Question 21

What are the two parts called that make up the gel matrix?

Answer 21

• The stacking gel

- The resolving gel

Question 22

What are the concentrations in the stacking gel?

Answer 22

5% acrylamide
Tris/HCl pH6.9

• Glycinate low mobility
• Cl- high mobility

Question 23

What is happening in the stacking gel?

Answer 23

• proteins move freely

- proteins stacked

Question 24

What are the concentrations in the resolving gel?

Answer 24

5-20% acrylamide
Tris/HCl pH 8 to 9

High [glycinate]
High [acrylamide]

Question 25

What happens to the proteins in the resolving gel?

Answer 25

proteins separated by size/shape and charge

Question 26

What are the two types of PAGE gels you can get?

Answer 26

- Non- denaturing Native PAGE | - SDS PAGE

Question 27

How can Native PAGE separate proteins?

Answer 27

size, charge and shape

Question 28

What are the advantages of Native PAGE?

Answer 28

- Retains quaternary structure activity - Detect with coloured substrate

Question 29

What are the disadvantages of Native PAGE?

Answer 29

- Aggregates block | - Interpretation difficult

Question 30

How can the molecular mass be determined?

Answer 30

- Use a Ferguson plot -

Question 31

How can you plot and you a Ferguson plot?

Answer 31

``` Mobility (v/E)  1/h  1/(% gel monomer) Plot log(relative mobility) vs % gel ```

Question 32

What does SDS stand for?

Answer 32

Sodium Dodecyl Sulphate

Question 33

What is an advantage of SDS PAGE?

Answer 33

- removes aggregates

Question 34

How does SDS page work?

Answer 34

- Intercalates hydrophobic core unfolds - Boil to increase denaturation - Coats with negative charge - Constant charge : size ratio - Constant rod shape

Question 35

How does SDS intercalate into the protein core?

Answer 35

The deteregent part is looking for a hyrophobic environmnet , goes into into the cnetre of the protein and helps it underfold

Question 36

What do you need to add to SDS page ?

Answer 36

``` Bromophenol blue (shows gel front) b-mercaptoethanol (reduce S=S) Molecular weight marker proteins ```

Question 37

What are the uses of SDS page?

Answer 37

Assess purity | Estimate molecular weight

Question 38

How can you determine the Molecular weight from SDS page?

Answer 38

- Using proteins of known MW - mobility vs log(MW) - Determine unknown MW

Question 39

What is the equation for characteristic mobility ?

Answer 39

Top of resolving gel to protein band/ | Top of resolving gel dye front

Question 40

What is a western blot?

Answer 40

specific protein detection using antibodies

Question 41

What are the 5 steps that are done to do a Western blot?

Answer 41

1. Electroblot perpendicular to SDS direction 2. Block non- specific sites 3. Add specific antibody 4. Bind secondary antibody 5. Detect with colour product or radiojlabel

Question 42

What is Isoelectric focussing?

Answer 42

Focussing proteins at individual pI | Electrophoresis in a stable pH gradient

Question 43

What does the gel contain in Isoelectric focussing?

Answer 43

Ampholytes (+H3N)a-(CH2)n-(COO-)

Question 44

How does isoelectric focussing work?

Answer 44

proteins migrate to where their Pi = pH , they stop here as there is no net charge and so they no longer move

Question 45

What are the uses of Isoelectric focussing?

Answer 45

- Detection of phosphorylation | - or isoenzymes in diagnosis and forensics

Question 46

What are the differences between 1D vs 2D gel electrophoresis s?

Answer 46

1D GE separates < 100 proteins 2D gel electrophoresis (2DGE) IEF in gel strip (from low to high) SDS-PAGE