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Electrophoresis

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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Electrophoresis

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  1. 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.

  2. Electrophoresis Equipment for electrophoresis is a power pack and an electrophoresis unit (gel tank) – either vertical or horizontal. Images from Anachem Ltd

  3. Methodology of SDS-PAGE gels

  4. *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 classifyingpolypeptides. * The best concentration for particular size ranges has thankfully been determined by previous scientists. Polyacrylamide Gels (i.e. SDS-PAGE)

  5. Gels for Separating Proteins

  6. 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

  7. Mixture of polypeptides of known molecular weights 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 weight Molecular Weight Standards

  8. Mixture is heated in a boiling water bath for a few minutes to denature the proteins Sample preparation for SDS-PAGE Protein samples are suspended in a buffer solutioncontaining SDS, -mercaptoethanol, glycerol and a tracking dye • 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

  9. SDS-Page Loading the gel Connecting to the power supply

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