Exp.5-1 Cellulose Acetate E

EXPERIMENT 3.1

SEPARATION AND CLASSIFICATION OF SERUM PROTEINS BY CELLULOSE ACETATE MEMBRANCE ELECTROPHORESIS

Objective Master the principle and method of electrophoresis to separate and identify serum proteins using cellulose acetate membrane as a supporting medium .

Principle In alkaline buffer solution, serum proteins are negatively charged . They will migrate toward anode in electric field. Because of the differences of q and molecular weight, their migration velocities are distinct. After electrophoresis, immerse cellulose acetate film into the dye solution. Then five zones of serum proteins can be seen after the film is stained and destained, including albumin, α 1-globulin, α 2-globulin β-globulin, and γ-globulin fraction.

Procedure 

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Immerse a cellulose acetate membrane into the electrophoresis buffer, take it out and absorb water with filter paper. Add 3~5μl sample on the rough side of membrane at the site 2 cm from cathode, and put the membrane on the electrophoresis device with the rough side of membrane down, connect the electrode and the film with filter paper. Electrophoresis is carried out at a constant voltage of 110V for 50~60 min. After electrophoresis, take out membrane, immerse into the dye (Amino Black-10B ) solution for 1 min .Then put it into destaining solution for rinsing. Observe and judge the shade of color and width of the bands to estimate their quantity.

Reference 

Serum Protein Electrophoresis--Normal Pattern

Separated protein bands are visualized in characteristic positions after being staining

Clinical significance The concentration of total proteins in the plasma is about 60-80 g/L. The plasma proteins are a complicated mixture that includes not only simple proteins but also mixed or conjugated proteins (glycoproteins and lipoproteins). The Cellulose acetate membrane electrophoresis can separate the plasma proteins into 5 fractions. Albumin is the major protein of human plasma (3848g/L,nearly 50% of the total plasma proteins. The liver produces about 12 g of albumin per day.

Albumin has two main functions: (1) maintaining colloid osmotic pressure of blood; (2) transportation: acting as a carrier molecule for bilirubin, fatty acids, trace elements and many drugs. As an indicator of liver and kidney disease, immune deficiencies, malignancies of the immune system, acute and chronic infection, genetic deficiencies, central nervous system disease and numerous other pathologies, serum protein electrophoresis exceeds any other single test in its ability to detect so broad a spectrum of disease states.