MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

1. MLAB 2401: Clinical ChemistryKeri Brophy-Martinez Electrophoresis

2. Electrophoresis • Principle • the migration of charged solutes or particles in a liquid medium under the influence of an electrical field. • Distance traveled by the particle depends on its molecular weight and overall charge • Types • Iontophoresis • Migration of ions • Zone electrophoresis • Migration of macromolecules

3. Electrophoresis • Clinical Application • Proteins • Immunoglobulins • Hemoglobin • Isoenzyme/enzyme • Lipoprotein

4. Components • Driving force/ electrical power • Support medium • Buffer • Sample • Detecting system + Anode = Cathode

5. Support Mediums • Cellulose Acetate • Dry and brittle • Becomes pliable when soaked in electrolyte buffer • After electrophoresis, it can be stained and read in a densitometer • Long term storage possible

6. Support Mediums • Agarose Gel • Purified agar • No electroendosmosis • After electrophoresis, it can be stained and read in a densitometer • Long term storage possible • Small sample size ~ 2-10 µL required

7. Support Mediums • Polyacrylamide Gel • Gels with different pore sizes can be layered to provide good separation of molecules of different sizes • Good resolution • Detect 20 serum protein fraction rather than 5

8. Procedure • Serum is applied to the support media and the protein dissolves in the buffer, giving them an electric charge • A specific amount of current is applied for a specific amount of time • As the current flows through the media, the electrically charged molecules migrate along the supporting media

9. Procedure • The negatively charged protein molecules migrate towards the oppositely charged electrode • The sample is separated into bands where each band has molecules containing similar mobility

10. Staining of the Supporting Medium • Staining fixes the protein to the membrane by denaturing • Makes the fractions visible • Decolorization is used to remove background color • Each peak in each column represents a different band of molecules that migrated together

11. Densitometer • A densitometer scans the stained strip and reports a graphical representation of the bands

12. Densitometer • As the light beam passes through each stained band, the percent transmission is recorded and a graph representation of the concentration is recorded • A decrease in % T means the concentration of the fraction is increased and seen as a large peak on the scan • An increase in %T is graphed as a low peak or no peak • Each protein fraction can be calculated by determining the area

13. Factors Affecting Migration Rates • Molecular weight/ size/shape • Molecular charge in the buffer • Net charge of particles • Type of supporting medium • Temperature • Electrical voltage • Migration time

14. Protein Electrophoresis + =

15. Relative Percent of Protein Bands

16. Common Electrophoresis Patterns

17. More Electrophoresis Patterns Jarreau, P. (2005). Clinical Laboratory Science review (3rd ed.). New Orleans, LA: Louisiana State University Health Science Center Foundation.

18. Hemoglobin Electrophoresis • Principle and system is the same as protein electrophoresis • Solubility is an important factor in the mobility of the hemoglobin proteins

19. Hemoglobin Electrophoreis Patterns

20. References • Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. • http://www.funsci.com/fun3_en/exper1/exper1.htm • http://themedicalbiochemistrypage.org/hemoglobin-myoglobin.html • http://science-project.com/OnlineCatalog.html • Jarreau, P. (2005). Clinical Laboratory Science review (3rd ed.). New Orleans, LA: Louisiana State University Health Science Center Foundation. • Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .