Electrophoresis

• Defined as the migration of charged particles through a solution under the influence of an electric field.

• Many important biological molecules possess ionisable groups– e.g. amino acids, peptides, proteins,

nucleotides, nucleic acids• So, at a given pH they exist in solution as

electrically charged species either as cations (+) or anions (-).

• If an electric field is applied charged particles will either migrate to the cathode or anode depending upon their charge.

Electrophoresis

Equipment for electrophoresis is a power pack and an electrophoresis unit (gel tank) – either vertical or horizontal.

Images from Anachem Ltd

Methodology of SDS-PAGE gels

Polyacrylamide Gels (i.e. SDS-PAGE)* Stacking gel (4.5%)

– Stacks all the polypeptides into a narrow band – Allows all the polypeptides to enter the separating gel at the same time

* Separating gel (10-12%)– Separates the various polypeptides based on their molecular weight– The smaller the polypeptides the faster it will migrate

• The smaller the polypeptides size, the higher acrylamide concentration needed to properly separate.

* Otherwise, the small proteins would just race through the gel matrix with no quantitative results for classifying polypeptides.

* The best concentration for particular size ranges has thankfully been determined by previous scientists.

Gels for Separating Proteins

Using a stacking gel

low percentacrylamide (4%)

Resolution of good bands in resolving gel relies on all the sample entering the gel at the same time

Molecular Weight Standards1. Mixture of polypeptides of known molecular

weights2. Helps to determine the molecular weight of

an unknown polypeptide3. Does not tell you what proteins or

polypeptides are in your sample(a) Because two proteins have the same molecular weight does not mean they are the same protein(b) May be hundreds of different proteins with the same molecular weight

Sample preparation for SDS-PAGE

Mixture is heated in a boiling water bath for a few minutes to denature the proteins

Protein samples are suspended in a buffer solutioncontaining SDS, -mercaptoethanol, glycerol and a

tracking dye

(a) Must have excess SDS (at least 3:1 ratio)

(b) Must have excess reducing agent

Even when you take all precautions you must still be carefulwhen interpreting your results

SDS-Page

Loading the gel Connecting to the power supply