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BB10186: Proteins purification and characterisation > lecture 10 - electrophoresis > Flashcards

lecture 10 - electrophoresis Flashcards

1
Q

How does gel electrophoresis work?

A
  • charged proteins separate in an electric field
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2
Q

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

A
  • Feels an attractive force towards the electrode

- FE= Fq

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3
Q

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

A
  • protein size
  • velocity
  • viscosity
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4
Q

what is stokes law ?

A

Velocity = 6phr

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5
Q

What does Stokes law depend on?

A
  • the size of the protein
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6
Q

What is the apparatus needed for the electrophoresis ?

A
  • pair of platinum electrodes on either side of the buffer reservoir , into which is placed a solid supporting medium which may be inert
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7
Q

why do we have to use a buffer?

A

to keep the pH the same

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8
Q

What happens when the pH = pI?

A

the protein will not move , the charges are the same

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9
Q

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

A
  • minimize convection and denaturation
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10
Q

Why two types of solid supporting matrixes can you have?

A
  • horizontal or vertical
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11
Q

What are 2 factors that the matrix must be?

A
  • inert

- porous

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12
Q

Why must the matrix be inert?

A
  • the protein does not interact with it
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13
Q

Why must the matrix be porous?

A

allow separation for charge and size

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14
Q

What is the most common gel matrix used?

A

polyacrylamide, (agarose, if protein is very large)

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15
Q

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

A
  • Coomassie Brilliant Blue
  • Silver Stain
  • Specific detection
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16
Q

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

A
  • In MeOH/CH3COOH to denature & fix
  • Destain in same without dye
  • Detects 1mg-100ng
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17
Q

What volume can silver stain detect?

A

1ng

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18
Q

What does PAGE stand for?

A

Polyacrylamide Gel Electrophoresis

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19
Q

How is the PAGE gel formed?

A

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

20
Q

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

A
  • depends on the concentration of the acrylamide
21
Q

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

A
  • The stacking gel

- The resolving gel

22
Q

What are the concentrations in the stacking gel?

A

5% acrylamide
Tris/HCl pH6.9

  • Glycinate low mobility
  • Cl- high mobility
23
Q

What is happening in the stacking gel?

A
  • proteins move freely

- proteins stacked

24
Q

What are the concentrations in the resolving gel?

A

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

High [glycinate]
High [acrylamide]

25
Q

What happens to the proteins in the resolving gel?

A

proteins separated by size/shape and charge

26
Q

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

A
  • Non- denaturing Native PAGE

- SDS PAGE

27
Q

How can Native PAGE separate proteins?

A

size, charge and shape

28
Q

What are the advantages of Native PAGE?

A
  • Retains quaternary structure
    activity
  • Detect with coloured substrate
29
Q

What are the disadvantages of Native PAGE?

A
  • Aggregates block

- Interpretation difficult

30
Q

How can the molecular mass be determined?

A
  • Use a Ferguson plot

-

31
Q

How can you plot and you a Ferguson plot?

A 
Mobility (v/E)  1/h  1/(% gel monomer)
Plot log(relative mobility) vs % gel
32
Q

What does SDS stand for?

A

Sodium Dodecyl Sulphate

33
Q

What is an advantage of SDS PAGE?

A
  • removes aggregates
34
Q

How does SDS page work?

A
  • Intercalates hydrophobic core unfolds
  • Boil to increase denaturation
  • Coats with negative charge
  • Constant charge : size ratio
  • Constant rod shape
35
Q

How does SDS intercalate into the protein core?

A

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

36
Q

What do you need to add to SDS page ?

A 
Bromophenol blue (shows gel front)
 b-mercaptoethanol (reduce S=S)
Molecular weight marker proteins
37
Q

What are the uses of SDS page?

A

Assess purity

Estimate molecular weight

38
Q

How can you determine the Molecular weight from SDS page?

A
  • Using proteins of known MW
  • mobility vs log(MW)
  • Determine unknown MW
39
Q

What is the equation for characteristic mobility ?

A

Top of resolving gel to protein band/

Top of resolving gel dye front

40
Q

What is a western blot?

A

specific protein detection using antibodies

41
Q

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

A
  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
42
Q

What is Isoelectric focussing?

A

Focussing proteins at individual pI

Electrophoresis in a stable pH gradient

43
Q

What does the gel contain in Isoelectric focussing?

A

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

44
Q

How does isoelectric focussing work?

A

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

45
Q

What are the uses of Isoelectric focussing?

A
  • Detection of phosphorylation

- or isoenzymes in diagnosis and forensics

46
Q

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

A

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

BB10186: Proteins purification and characterisation (10 decks)

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