Immunoelectrophoresis experiment of polyclonal antibody

Immunoelectrophoresis experiment of polyclonal antibody

Immunoelectrophoresis experiment of polyclonal antibody
[Experimental principle]
Immunoelectrophoresis, also known as gamma globulin electrophoresis or immunoglobulin electrophoresis, is an experimental method that can determine the level of three immunoglobulin IgM'' IgG'' IgA levels in the blood. In this experimental technique, the protein is first separated by horizontal agarose gel electrophoresis using the molecular weight of the protein and the ratio of the charged charge, and then the specific antibody is introduced into the groove parallel to the electrophoresis direction in the antigen. During the diffusion of antibodies and antibodies, the proper ratio of antigen to antibody will result in the production of precipitates (Figure). The 0.85% salt can not only terminate the diffusion process but also wash away the unbound protein. The precipitation line formed by the combination of the antigen and the antibody can be observed by the naked eye or by the dyeing 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 screening of immune complexes, identification and identification of various abnormal gamma globulinemia. Immunoelectrophoresis is also a reliable and accurate method for routine protein evaluation, which can observe changes in protein structure and concentration.
【Experimental Materials】
1. Experimental equipment level electrophoresis apparatus; puncher; dropper; blade; power supply; water bath (55 ° C); distilled water; beaker (400-600 ml);
Load the gun (5-100ul).
2. Experimental reagents (1) Purified antibody; protein mixture; 1% agar.
(2) Tris-barbital buffer solution: 2.24 g of barbituric acid, 4.43 g of Tris, 0.053 g of calcium lactate and 0.065 g of sodium azide dissolved in 100 ml of distilled water.
(3) Electrophoresis buffer solution: Mix Tris-barbital buffer solution with distilled water in a ratio of 1:4.
【experimental method】
1. Prepare the agarose gel. Mix the agar and the electrophoresis buffer solution into a 90 ° C water bath to heat to dissolve, make 1% agar, spread the agar on a horizontal glass plate before cooling, and then cool to room temperature. A punch with an inner diameter of 4 mm punches and makes a groove.
2. Electrophoresis 4%-5% of the antigen diluted with the electrophoresis buffer solution is carefully placed into the small hole with a dropper, the rubber plate is placed in the electrophoresis tank, and the buffer plate in the electrophoresis tank is connected with the wet filter paper. . Cover the electrophoresis tank cover, turn on the power, and move 6V/cm to the leading edge to move about 35mm for about 1 hour. Use the tracer dye to judge the moving distance.
3. Diffusion remove the rubber plate out of the electrophoresis tank and place it in a horizontal position. 'Use the filter paper to dry the buffer solution in the groove of the rubber plate'. Add an appropriate amount of antibody (0.2ml~0.25ml) to the groove, and note that the antibody cannot overflow. Grooves to avoid contamination. Move the rubber sheet to a box containing sodium azide and have a certain humidity. After capping and sealing in a 30 ° C water bath for at least 10 hours, move to 0.85% brine to stop the diffusion. Wash away unbound protein.
[Experimental results]
The precipitation line formed between the antigen and the antibody was observed and recorded.

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