Electrophoresis 2

Question 1

How cans separation occur with electrophoresis?

Answer 1

separation based on size only
- SDS PAGE = sodium dodecyl sulphate PAGE

separation based on charge only
- isoelectric focusing

Question 2

How SDS PAGE separation take place?

Answer 2

sample is heated and placed in a buffer containing

• 1% SDS
• 1% 2-mercaptoethanol = cleaves any disulphide bonds

results in polypeptide subunits that bind to SDS in a constant weight ratio
- creates uniform charge per unit length area
= any intrinsic charge on the polypeptide is swamped by SDS negative charge

uses of a gel of an appropriate pore size allows separation based on size due to the sieving effect

Question 3

How can we find the molecular weight/size of unknown polypeptides?

Answer 3

run calibration polypeptides of known molecular weight

- allows the molecular weights of unknown polypeptides

Question 4

How can Rf value be used to calculate molecular weight of polypeptides?

Answer 4

measure the migration distance

calculate the Rf values using the sample wells as the origin and the end of the gel as the reference point.

construct a standard curve of logMWt versus Rf for the known proteins.

use the standard curve to calculate
the MWt of the unknown

Question 5

What is isoelectric focusing?

Answer 5

high resolution method where proteins are separated in the presence of a continuous pH gradient
- proteins migrate according to their charge until they reach pH values at which they have no net charge

= the proteins will therefore attain a steady state of zero migration and will be concentrated in bands/sharp zones

Question 6

What is the isoelectric point?

Answer 6

it is the pH at which the proteins have no net charge and stop moving

Question 7

What is 2D separations?

Answer 7

used when you have proteins with similar molecular weights or isoelectric points
- means SDS PAGE or isoelectric focusing alone will not separate them