ELECTROPHORESIS FRACTIONATION Medical Biochemistry Department By All Staff Members of Medical Biochemistry Department 2011-2012 ILO of the current topic: By the end of this topic, the student will be able to: Interpret electrophoresis results for serum proteins and hemoglobin.

ELECTROPHORESIS FRACTIONATION Definition Electrophoresis is the migration of charged particles such as proteins dissolved in a buffer on, or in, a supporting medium when an electric current is passed through them. gel electrophoresis used to separate macromolecules as proteins and nucleic acids

Principle of Separation According to charge According to size

(1) According to charge When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.

(2) According to size

Instruments and Reagents Power supply Tank ( Buffer ,Supporting media) Detection and Quantification Cathod e Anode Electrophoresis tank Paper support medium

Additional accessories may be required for gel electrophoresis as combs, glass plates with their separators. paper electrophoresis apparatus Gel electrophoresis apparatus

Factors affecting the rate of migration of charged molecules: Size shape Electric current intensity Buffer pH Supporting media Nature Pore size

Electrophoresis pattern of normal serum proteins 2 3 4 5 Alb 1 2   Densitometer scanning from cellulose acetate strip.

Interpretation of the results: Albumin is the fastest protein , forming the largest band , and is closest to the positive electrode. The next 4 bands are globulins; alpha 1, apha 2, beta and gamma globulins, respectively.

Serum Protein Electrophoresis is useful in detecting: clinical disorders . Albumin Hypoalbuminemia Liver disease Kidney disease  1 – antitrypsin deficiency Immunoglobins Hypogammaglobinemia Hypergammaglobinemia

Interpretation of results require knowledge of common patterns Interpretation of results require knowledge of common patterns. You should always interpret according to the normal pattern Normal Pattern

Monoclonal Gammopathy Pattern The large spike in the gamma region is indicative of an increase in one immunoglobulin class typically seen in monoclonal gammopathies such as multiple myeloma.

-1 Antitrypsin Deficiency 1-antitrypsin deficiency appears as either a decrease or complete absence of the 1 band depending on whether the condition is heterozygous or homozygous.

Nephrotic Syndrome The nephrotic syndroma pattern is characterized by decreases in all protein fractions except -2. -2- Macroglobulin is retained by the kidney owing to its large molecular size.

Liver Cirrhosis The typical electrophoresis pattern of liver cirrhosis shows a merging of the  and  fraction, known as the - bridge

Lab activities 1-You are provided with a protein sample ,estimate total serum protein with photometer 2-Interpret the results of your sample according to electrophoresis diagram

Calculation of the results Determination of the total serum proteins concentration Total serum proteins (g/dl) =AT/AS X conc. of standard- Absolute concentration (gm/dl) of protein fractions: Absolute concentration of a fraction = P/100 X C. Where P = the percent of each zone and C = the total serum protein concentration. In gm/dl.

Absolute Concentration (gm/dl) Interpretation of results: Normal serum protein electrophoresis using cellulose acetate paper and by elution gives a pattern in which the various protein fractions as follows: Protein Fraction Percentage (%) Absolute Concentration (gm/dl) 1- Albumin 55 –65% 3.5 – 5.0 2- 1 Globulins 1.0 –5% 0.17 – 0.33 3- 2 globulins 4.9 –11.2% 0.42 – 0.90 4-  Globulins 7 – 13% 0.52 – 1.05 5-  Globulins 9.8 – 18.2% 0.71 – 1.65