Rocket electrophoresis experiment

Summary

A fixed amount of antigen is added to the agarose gel containing a certain amount of the corresponding antibody, under the action of the electric field, causing the migration and mutual reaction of the antigen and antibody, when the proportion is appropriate to form the precipitation peak visible to the naked eye, due to the electrophoresis continues to be carried out, the sample wells continue to have the antigen to move to the antigen-antibody complex precipitation peaks, due to the excess of the antigen so that the precipitation is dissolved in front of the precipitation peaks in the formation of new antigen-antibody complexes.

Operation method

Rocket electrophoresis

Principle

Under the action of the electric field, the antigen swims in the agar medium containing quantitative antibody, and the ratio of the two is suitable to form a precipitation line resembling a rocket or a cone in a short time, and the height of this precipitation line is often proportional to the amount of antigen, so this method can determine the amount of antigen in the sample.

Materials and Instruments

Serum to be tested
Barbiturate buffer Agar powder
Micro-sampler Perforator Glass plate Electrophoresis apparatus

Move

1. Preparation of antibody agar plates: Same as one-way diffusion method, but note that dilutions should be made with 0.05 M barbiturate buffer pH 8.6 ionic strength.
2. Perforation is shown in the figure below Fig. 1 Rocket electrophoresis antigen well location map3. Add the appropriate concentration of reference serum diluted with buffer and the appropriate dilution of antigen (human serum) into each well, 10 or 20 microliters per well, requiring accurate addition without spillage.
4. Put the spiked immunoagar plate into the electrophoresis tank for electrophoresis at 4-6 volts/cm, electrophoresis time 1-5 hours, until most of the antigen wells appear in front of the tip of the narrow and completely closed rocket-shaped precipitation line, turn off the power.
5. Remove the agar plate, take the center of the antigen wells as the starting point and measure the height of each rocket-shaped precipitation line. Plot the standard curve in the same way as the unidirectional agar diffusion method to find out the Ig content in the serum to be tested.

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