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Protein Electrophoresis

Protein Electrophoresis. Chapter 10 Electrophoretic techniques. Electrophoresis. Method where charged molecules in solution, eg proteins and nucleic acids, migrate in response to electrical field within a gel. Migration rate depends on: the strength of the field on the net charge

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Protein Electrophoresis

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  1. Protein Electrophoresis Chapter 10 Electrophoretic techniques

  2. Electrophoresis Method where charged molecules in solution, eg proteins and nucleic acids, migrate in response to electrical field within a gel. • Migration rate depends on: • the strength of the field • on the net charge • size, and shape of the molecules • the ionic strength, viscosity, and temperature of the medium in which the molecules are moving

  3. Net charge is determined by the pH of the medium • Proteins are amphoteric compounds, that is, they contain both acidic and basic residues • Each protein has its own characteristic charge properties depending on the number and kinds of amino acids carrying amino or carboxyl groups • Nucleic acids, unlike proteins, are not amphoteric. They remain negative at any pH used for electrophoresis

  4. In most electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is through the gel.

  5. Electrophoresis -vertical

  6. Electrophoresis in tubes

  7. Tube Gel Units

  8. Electrophoresis -horizontal

  9. Electrophoresis

  10. Interrelation of Resistance, Voltage, Current and Power • Two basic electrical equations are important in electrophoresis • The first is Ohm's Law, I = E/R • The second is P = EI • This can also be expressed as P = I2R • In electrophoresis, one electrical parameter, either current, voltage, or power, is always held constant

  11. Consequences • Under constant current conditions (velocity is directly proportional to current), the velocity of the molecules is maintained, but heat is generated. • Under constant voltage conditions, the velocity slows, but no additional heat is generated during the course of the run • Under constant power conditions, the velocity slows but heating is kept constant

  12. Electrophoresis • Powerpack supplies current between the electrodes in the electrophoresis unit. • Electrophoresis is carried out in appropriate buffer to maintain • ionisation of separating molecules. • Read page 449-453 regarding mobility of molecules in a potential • gradient created in a gel. • Find out: • how to calculate the velocity of a charged molecule in an electric field. • what the term electrophoretic mobility means. • How to increase mobility by increasing current or voltage • and the draw-backs of this. • 4. About the effects of electroosmosis flow

  13. Electrophoresis cellulose acetate

  14. Electrophoresis cellulose acetate Useful for qualitative and semi-quantitative analysis…It is rapid.

  15. Electrophoresis Agarose gels

  16. Electrophoresis Agarose gels

  17. Electrophoresis Agarose gels Used mainly for large proteins and nucleic acids (eg RNA and DNA) due to the large pore sizes. The low melting temperature also allows bands to be cut out of the gel, the gel melted and the sample recovered easily. Also useful immunodiffusion since it allows large proteins to diffuse through the gel unhindered.

  18. Agarose gelsLab 4(2) Separation of LDH isoenzymes Isoenzymes = different molecular forms of the same enzyme. Five major LDH isoenzymes are found in different vertebrate tissues. There are two types of polypeptide chains in LDH: M (for skeletal muscle)…pyruvate to lactate H (for heart muscle) ……pyruvate to citric acid These are combined into the LDH tetramer in 5 different ways. The electrophoretic mobilities of the LDH isoenzymes are: LDH 1 > LDH 2 > LDH 3 > LDH 4 > LDH 5.

  19. Agarose gelsLab 4(2) Separation of LDH isoenzymes In a myocardial infarction, enhanced LDH5 level is proportional to the damage of the heart muscle tissue In serious cases 3X elevation in the plasma The elevation of serum LDH activity might represent other diseases (e.g. anemia, tumors, liver diseases) as well, so it is important to know from what tissue the LDH was released into the bloodstream.

  20. Agarose gelsLab 4(2) Separation of LDH isoenzymes LDH activity is easily detected using ENZYME ACTIVITY of LDH

  21. Agarose gelsLab 4(2) Separation of LDH isoenzymes PMS = artificial acceptor

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