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
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Equipment for electrophoresis is a power pack and an electrophoresis unit (gel tank) – either vertical or horizontal.
Images from Anachem Ltd
*Stacking gel (4.5%)
Stacks all the polypeptides into a narrow band
Allows all the polypeptides to enter the separating gel at the
* 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 classifyingpolypeptides.
* The best concentration for particular size ranges has thankfully been determined by previous scientists.Polyacrylamide Gels (i.e. SDS-PAGE)
low percentacrylamide (4%)Using a stacking gel
Resolution of good bands in resolving gel relies on all the sample entering the gel at the same time
Helps to determine the molecular weight of an unknown polypeptide
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 weightMolecular Weight Standards
Mixture is heated in a boiling water bath for a few minutes to denature the proteinsSample preparation for SDS-PAGE
Protein samples are suspended in a buffer solutioncontaining SDS, -mercaptoethanol, glycerol and a
(b) Must have excess reducing agent
Even when you take all precautions you must still be carefulwhen interpreting your results
Loading the gel
Connecting to the power supply