Electrophoretic Techniques Flashcards
What are the uses for SDS-PAGE?
Determine protein size Identify protein Determine sample purity Identify existence of disulfide bonds Quantify amounts of protein SDS is an anionic detergent.
What does SDS-PAGE stand for?
Sodium dodecyl sulphate polyacrylamide gel electrophoresis.
How are polyacrylamide gels formed?
Cross linked polyacrylamide gels are formed from the polymerisation of acrylamide monomer in the presence of smaller amounts of bis-acrylamide (cross linking agent).
What is western blotting?
Combines resolution of SDS-PAGE with specificity of antibody detection.
What are the uses for western blotting?
Presence of an antigen
Amount of an antigen present
Molecular weight of antigen
Post-translational modifications (e.g. phosphorylation)
What are the steps for western blotting?
1) Proteins are loaded and electrophoresed onto a PAGE.
2) Proteins are then transferred onto nitro-cellulose membrane, followed by incubation with 5% dried milk powder to block no specific binding.
3) the nitro-cellulose membrane is then incubated with the primary antibody.
4) after series of washes the membrane is probed with secondary antibody or fluorescent.
5) the bands are visualised using a chemilluminescence or fluorescence detector and the MW of visible bands is estimated.
What are the steps for Iso-electric Focussing? (IEF)
Protein mixture is absorbed onto gel. Electric field is then applied across gel.
Proteins in regions below or above their pI migrate to the cathode or anode respectively.
Protein migration ceases when pH=pI.
What is 2D gel electrophoresis?
Combines IEF and SDS-PAGE separations into a single gel slab.
The combination of these two techniques provides a highly sophisticated analytical method for analysing protein mixtures.
What is electrophoresis?
The migration of charged particles under the influence of a direct electric current.
Why is it necessary to have small pores in the gel slab?
So that small fragments can move faster than the bigger ones.
Why is the cross linking agent used to make polyacrylamide gels?
It has more effect on elasticity where the pore size of the gel decreases with the increasing proportion of bis-acrylamide.
Why do we need TEMED (tetramethylethylene)?
Because an initiator is required to start the reaction.
Why is PAGE required?
In DNA sequencing it provides a fundamental method of separation.
What is iso-electric focussing?
A molecule will not move in an electric field if it’s at it’s iso-electric pH (pI). If electrophoresis is performed in an electrolyte that has a pH gradient, molecules will migrate to the point where the pH of the solution is the same as their iso-electric pH and remain there as long as the gradient and potential differences are maintained.
In IEF what pH is the cathode and anode?
An acid is used at the anode (+) and an alkali at the cathode (-).
