lecture 10 - electrophoresis Flashcards
How does gel electrophoresis work?
- charged proteins separate in an electric field
What does the protein of charge q feel in the electric field E
- Feels an attractive force towards the electrode
- FE= Fq
What is the frictional force F opposed to and proportional to?
- protein size
- velocity
- viscosity
what is stokes law ?
Velocity = 6phr
What does Stokes law depend on?
- the size of the protein
What is the apparatus needed for the electrophoresis ?
- pair of platinum electrodes on either side of the buffer reservoir , into which is placed a solid supporting medium which may be inert
why do we have to use a buffer?
to keep the pH the same
What happens when the pH = pI?
the protein will not move , the charges are the same
Why must the solid supporting matrix of the apparatus be thin?
- minimize convection and denaturation
Why two types of solid supporting matrixes can you have?
- horizontal or vertical
What are 2 factors that the matrix must be?
- inert
- porous
Why must the matrix be inert?
- the protein does not interact with it
Why must the matrix be porous?
allow separation for charge and size
What is the most common gel matrix used?
polyacrylamide, (agarose, if protein is very large)
What are the 3 ways you can detect protein on a gel matrix?
- Coomassie Brilliant Blue
- Silver Stain
- Specific detection
what are the chemicals used for Coomassie Brilliant blue and what are the measurement it can detect?
- In MeOH/CH3COOH to denature & fix
- Destain in same without dye
- Detects 1mg-100ng
What volume can silver stain detect?
1ng
What does PAGE stand for?
Polyacrylamide Gel Electrophoresis
How is the PAGE gel formed?
Acrylamide polymer
with N,N’-methylene bisacrylamide crosslinks,
Catalysed by SO4-•
How big can the pore size be in the PAGE gel?
- depends on the concentration of the acrylamide
What are the two parts called that make up the gel matrix?
- The stacking gel
- The resolving gel
What are the concentrations in the stacking gel?
5% acrylamide
Tris/HCl pH6.9
- Glycinate low mobility
- Cl- high mobility
What is happening in the stacking gel?
- proteins move freely
- proteins stacked
What are the concentrations in the resolving gel?
5-20% acrylamide
Tris/HCl pH 8 to 9
High [glycinate]
High [acrylamide]
What happens to the proteins in the resolving gel?
proteins separated by size/shape and charge
What are the two types of PAGE gels you can get?
- Non- denaturing Native PAGE
- SDS PAGE
How can Native PAGE separate proteins?
size, charge and shape
What are the advantages of Native PAGE?
- Retains quaternary structure
activity - Detect with coloured substrate
What are the disadvantages of Native PAGE?
- Aggregates block
- Interpretation difficult
How can the molecular mass be determined?
- Use a Ferguson plot
-
How can you plot and you a Ferguson plot?
Mobility (v/E) 1/h 1/(% gel monomer) Plot log(relative mobility) vs % gel
What does SDS stand for?
Sodium Dodecyl Sulphate
What is an advantage of SDS PAGE?
- removes aggregates
How does SDS page work?
- Intercalates hydrophobic core unfolds
- Boil to increase denaturation
- Coats with negative charge
- Constant charge : size ratio
- Constant rod shape
How does SDS intercalate into the protein core?
The deteregent part is looking for a hyrophobic environmnet , goes into into the cnetre of the protein and helps it underfold
What do you need to add to SDS page ?
Bromophenol blue (shows gel front) b-mercaptoethanol (reduce S=S) Molecular weight marker proteins
What are the uses of SDS page?
Assess purity
Estimate molecular weight
How can you determine the Molecular weight from SDS page?
- Using proteins of known MW
- mobility vs log(MW)
- Determine unknown MW
What is the equation for characteristic mobility ?
Top of resolving gel to protein band/
Top of resolving gel dye front
What is a western blot?
specific protein detection using antibodies
What are the 5 steps that are done to do a Western blot?
- Electroblot perpendicular to SDS direction
- Block non- specific sites
- Add specific antibody
- Bind secondary antibody
- Detect with colour product or radiojlabel
What is Isoelectric focussing?
Focussing proteins at individual pI
Electrophoresis in a stable pH gradient
What does the gel contain in Isoelectric focussing?
Ampholytes (+H3N)a-(CH2)n-(COO-)
How does isoelectric focussing work?
proteins migrate to where their Pi = pH , they stop here as there is no net charge and so they no longer move
What are the uses of Isoelectric focussing?
- Detection of phosphorylation
- or isoenzymes in diagnosis and forensics
What are the differences between 1D vs 2D gel electrophoresis s?
1D GE separates < 100 proteins
2D gel electrophoresis (2DGE)
IEF in gel strip (from low to high)
SDS-PAGE
