Electrophoresis

Question 1

Agarose electrophoresis

Answer 1

Uses 0.5%-2% agarose for separation of DNA/RNA

Horizontal gel

Utilises the native structure of molecules; molecules migrate towards the negative electrode

Loading dyes = bromophenol blue, xylene

Question 2

SDS-PAGE

Answer 2

Commonly used for separating proteins

Vertical gel

Uses the molecular weight of denatured proteins for separation; molecules migrate towards the positive electrode

Loading dye= bromophenol blue

Stains = commassie blue

Question 3

Preparation for SDS-PAGE

Answer 3

The protein is first boiled to linearize the polypeptide chain

SDS is then added and binds at a fixed ratio to the polypeptide chain

DDT/B-mercaptoethanol is then added which breaks sulphide bonds, allowing for complete linearization

Question 4

SDS

Answer 4

Sodium dodecyl sulphate

An anionic detergent that binds to polypeptides at a very fixed ratio, hence proteins can be separated based on the amount of SDS molecules bound

Has a negative charge and so causes the proteins to move towards the positive electrode

Question 5

Polyacrylamide

Answer 5

SDS-PAGE is carried out on a polyacrylamide gel

The gel is synthesised from acrylamide and BIS

The reaction is catalysed by persulfate and TEMED

Question 6

Discontinuous buffer system

Answer 6

SDS-PAGE uses a discontinuous buffer system; different areas of the gel are buffered to a different pH

• Stacking gel (pH 6.8) = This area focuses the sample into a narrow band
• Resolving gel (pH 8.8) = This area causes proteins to be separated by their molecular weight

Question 7

Western blotting

Answer 7

Proteins obtained from SDS-PAGE can be transferred onto a nylon membrane

The membrane is then probed using antibodies to identify specific proteins

Question 8

Isoelectric focussing

Answer 8

Used to determine the isoelectric point of a denatured protein

Achieved using a pH gradient; a band forms at charge neutrality