Week 11 (biochemical Methods II) 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?
agarose polyacrylamide
Describe the properties and functions of the gels
These gels form a mesh like structure, which acts like a molecular sieve
How do large and small molecules move through the mesh?
- large molecules move slowly through the mesh
- small molecules can move through the mesh easily
What can be altered to gives 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
Describe distance travels in relation to size of molecules
- Smallest molecules travel furthest
- Large molecules move less
What are the applications of electrophoresis?
Proteins DNA RNA
What are the analytical applications of electrophoresis?
- Check the purity of a sample
- Visualise the number of different molecules in a sample
- Estimate molecular mass
What is the main aim of electrophoresis?
Allows you to separate & visualise the components in your sample
Describe agarose gel electrophoresis for 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 Most DNA fragments are large
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 most be done to visualise the DNA use ethidium bromide?
-A fluorescent dye that intercalates with the DNA -Expose the gel to UV light
Describe SDS-PAGE for proteins
- Proteins can have a wide range of different charges -Proteins can have a wide range of different shapes
- Both of these can affect the velocity & migration of a protein in electrophoresis v = Ez/f To overcome this we use SDS-Poly-acrylamide Gel Electrophoresis
Explain SDS-PAGE for proteins: what is SDS?
Sodium dodecyl sulphate
Describe the properties of SDS page
-Is a detergent – denatures proteins
- loss of 2° & 3° structure
- only 1° structure remains
-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 (reducing agent) – break disulphide bonds
Heat sample Ensures denaturation occurs
Load on gel & apply voltage Include a lane of molecular weight standards
How can the components be visualised?
To visualise the proteins use a stain such as Coomassie Blue Initially whole gel goes blue Destain
– blue disappears from the regions where there is no protein
Explain the process for SDS-PAGE for proteins
Descrive the relationship between molecular weight and relative migration
