1. MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Electrophoresis

2. 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 Fraction% Albumin53-65 Alpha-1 globulin2-5 Alpha-2 globulin7-13 Beta globulin8-14 Gamma globulin12-20

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.