Experimental principle Immunoelectrophoresis, also known as γ-globulin electrophoresis or immunoglobulin electrophoresis, is an experimental method that can determine the levels of three immunoglobulins IgM, IgG, and IgA in the blood. In this experimental technique, first use the ratio of the molecular weight of the protein and the charge to separate the protein using horizontal agarose gel electrophoresis, and then introduce specific antibodies into the groove parallel to the direction of electrophoresis. During the diffusion process with the antibody, the proper ratio of antigen and antibody will cause precipitation. 0.85% salt can not only stop the diffusion process but also wash away unbound protein. The precipitation line formed by the combination of antigen and antibody can be observed either by naked eye or by staining method.
Immunoelectrophoresis technology can be used not only for the identification of monoclonal antibodies in serum or urine samples, but also for other aspects, such as the screening of immune complexes and the identification and identification of various abnormal gamma globulinemias. Immunoelectrophoresis technology is also a reliable and accurate experimental method for routine protein evaluation, which can observe changes in protein structure and changes in concentration.
Experimental reagent 1. Horizontal electrophoresis apparatus
2. Hole punch
3. Dropper
4. Blade
5. Power supply
6. Water bath (55 ℃)
7. Beaker (400-600ml)
8. Loading gun (5-100μl)
Experimental equipment 1. Purified antibody; protein mixture; 1% agar.
2. Tris-barbiturate buffer solution: 2.24g barbituric acid, 4.43g Tris, 0.053g calcium lactate and 0.065g sodium azide dissolved in 100ml distilled water.
3. Electrophoresis buffer solution: mix Tris-barbital buffer solution with distilled water at a ratio of 1: 4.
Experimental procedure 1. Prepare agarose gel
Mix the agar and the electrophoresis buffer solution into a 90 ° C water bath and heat to dissolve to make 1% agar. Spread the agar on a horizontal glass plate before cooling. After cooling to room temperature, use a 4 mm ID punch Punch and make grooves.
2. Electrophoresis
Place the 4% -5% antigen diluted with the electrophoresis buffer solution carefully into the small hole with a dropper, place the gel plate in the electrophoresis tank and connect the gel plate to the buffer solution in the electrophoresis tank with a wet filter paper. Cover the electrophoresis tank cover, turn on the power, and apply 6V / cm to the front edge to move about 35mm, the time is about 1 hour, use the tracer dye to judge the moving distance.
3. Diffusion
Move the gel plate out of the electrophoresis tank and place it in a horizontal position. Wet the buffer solution in the groove of the gel plate with filter paper, add an appropriate amount of antibody (0.2ml ~ 0.25ml) to the groove, pay attention to the antibody not to overflow the groove to avoid Contamination. Move the rubber plate to a box containing sodium azide and a certain humidity, cover and seal, and diffuse in a 30 ° C water bath for at least 10 hours. Move to 0.85% saline to stop the diffusion and wash off unbound protein. .
4. Observe and record the precipitation line formed between the antigen and antibody.
Matters needing attention 1. The success of immunoelectrophoresis analysis depends mainly on the quality of antiserum. The antiserum must contain enough antibody to generate a precipitation reaction with all the antigenic substances in the tested sample.
2. Although the antiserum contains corresponding antibodies to all antigenic substances, the antibody titer is high or low, so the size of the antigen pore size and the distance of the antibody groove should be properly considered.
3. Immunoelectrophoresis requires that the substance analyzed is an antigen and the other is a precipitation-reactive antibody. Therefore, there are no antigenic substances or substances with poor antigenicity and non-precipitating reactive antibodies, which cannot be analyzed by immunoelectrophoresis.
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