Week 12 (biochemical Methods III) Flashcards
Describe the theory of electrophoresis
-A molecule with a net charge will move in an electric field
- The speed (velocity, v) at which the molecule moves depends upon;
-the strength of the electric field, E
-the net charge on the molecule, z
-the frictional coefficient, f (how much drag it experiences)
v = Ez/f
Which gels is Electrophoresis is carried out in?
Polymer gels such as:
agarose
polyacrylamide
What is the purpose of gels in electrophoresis?
These gels form a mesh like structure, which acts like a molecular sieve
How do small/large molecules move through the mesh?
- small molecules can move through the mesh easily
- large molecules move slowly through the mesh
How can electrophoresis be altered to give the best resolution for the molecules you are interested in?
-Can alter the concentration (%) of agarose/acrylamide in the gel
need to choose the concentration that gives the best resolution for the molecules you are interested in
-Can also make gradient gels – from 20% acrylamide at the bottom to 4% acrylamide at the top – can increase the resolution
How far do large/ small molecules travel respectively?
- Smallest molecules travel furthest
- Large molecules move less
what are the applications of electrophoresis?
Proteins
DNA
RNA
What are the analytical applications of electrophoresis?
- Allows you to check the purity of samples
- Visualise the number of different molecules in a sample
- Estimate molecular mass
What is the main advantageous property of electrophoresis?
Allows you to separate & visualise the components in your sample
Explain Agarose gel electrophoresis based on the structure of DNA
- Phosphate groups in the backbone
- One charge per nucleotide, thus all fragments of DNA have the same ratio of charge to mass.
- Use agarose gels as these have larger pores (acrylamide pores are too small)
Explain the process of agarose gel electrophoresis for DNA?
-Form wells using a comb before the agarose sets
-Mix DNA with loading dye and then add to wells
Loading dye contains:
-Glycerol – dense, causes sample to sink in the wells
-Bromophenol blue – a dye, allows you to track progress as the dye moves through the gel at the front
- In one well load a DNA ladder
- A series of DNA fragments of known length
What is used to visualise the DNA?
- ethidium bromide
- A fluorescent dye that intercalates with the DNA
- Expose the gel to UV light
SDS-PAGE for proteins
Proteins can have a wide range of different charges and shapes
Proteins can have a wide range of different shapes
Both of these can affect the velocity & migration of a protein in electrophoresis
The shape of the protein can affect the velocity migration, how can this be overcome?
To overcome this we use SDS-Poly-acrylamide Gel Electrophoresis
What does SDS stand for?
Sodium dodecyl sulphate
What is SDS?
Is a detergent – denatures proteins
•loss of 2° & 3° structure
•only 1° structure remains
Is SDS positively charged?
What does the charge mean for the proteins?
No
Is negatively charged
•large number of SDS molecules bound to each protein means all proteins have a large negative charge overall
•roughly constant mass to charge ratio
Explain the process of SDS-PAGE for proteins
- Mix protein sample with Laemmli sample buffer
- glycerol – dense
bromophenol blue – tracker dye
SDS – denature protein & give it negative charge
β-me/DTT – break disulphide bonds - Heat sample
Ensures that full denaturation occurs
Not good for membrane proteins because it makes them aggregate - Load on gel & apply voltage
Include a lane of molecular weight standards
What can be done to visualise the proteins?
use a stain such as Coomassie Blue
Initially whole gel goes blue
Destain (contains the same solvents as the stain but none of the dye) – blue disappears from the regions where there is no protein
The staining is fairly quantitative
More protein= more stain
What is an alternative stain to Coomassie Blue?
silver stain – get black/brown staining of proteins
More sensitive than Coomassie- better for dilute samples
More expensive & time consuming than Coomassie
What is the number of negative charges in DNA directly proportional to?
The size of DNA
Where are the wells?
At the top
Biggest molecules at the top and the smallest at the bottom
What can be done to break disulphide bonds (in the tertiary structure)?
Add a reducing agent
Are SDS pages run vertically ?
Yes
What would a homodimer produce?
A single band
What would a heterodimer produce?
2 distance
What is the molecular migration
Distance from log to band
What do you do to find the molecular mass?
Add together the molecular weights of each band (kilo Daltons)
