MLAB 2401: Clinical ChemistryKeri Brophy-Martinez

Electrophoresis

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

Electrophoresis

• Clinical Application– Proteins– Immunoglobulins– Hemoglobin– Isoenzyme/enzyme– Lipoprotein

Components

• Driving force/ electrical power

• Support medium• Buffer• Sample• Detecting system

+ Anode

= Cathode

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

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

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

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

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

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

Densitometer

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

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

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

Protein Electrophoresis

+

=

Relative Percent of Protein Bands

Fraction %

Albumin 53-65

Alpha-1 globulin 2-5

Alpha-2 globulin 7-13

Beta globulin 8-14

Gamma globulin 12-20

Common Electrophoresis Patterns

More Electrophoresis Patterns

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

Hemoglobin Electrophoresis

• Principle and system is the same as protein electrophoresis

• Solubility is an important factor in the mobility of the hemoglobin proteins

Hemoglobin Electrophoreis Patterns

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 .