MLAB 2401: Clinical Chemistry
Keri 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
=
Cathode
+
Anode

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
Alpha-1 globulin
Alpha-2 globulin
Beta globulin
Gamma globulin
%
53-65
2-5
7-13
8-14
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 .