Enzyme immunoelectrophoresis can be applied to the analysis and identification of enzyme proteins.

Operation method

Enzyme immunoelectrophoresis

Principle

When antigen and antibody are electrophoresed in an agarose gel, the antigen is negatively charged and moves toward the positive pole under alkaline buffer conditions. Antigens of different molecular weights or with different charges move different distances under the same conditions. These antigens at different sites, and then with the relevant antibody by diffusion to form immunoprecipitation line. The antigen and antibody in enzyme immunoelectrophoresis are the enzyme and its corresponding antibody globulin. The immune complex formed by the enzyme antigen and the enzyme antibody is chromogenic with the substrate. The high sensitivity of the enzyme reaction makes enzyme immunoelectrophoresis a sensitive detection technique, but it is limited to the analysis and identification of enzyme proteins.

Materials and Instruments

Diseased leaves of plants infected with viruses
Rabbit Anti-Enzyme Antibody Agarose Sodium Barbiturate Hydrochloric Acid Buffer Hydrogen donor mixture
Electrophoresis apparatus Constant temperature box Perforator Enameling box

Move

1. Place a small glass rod in the center of a clean slide, and then pour 3 ml of dissolved agarose solution (0.8 g agarose/100 ml of sodium barbiturate hydrochloric acid buffer) on it, and when it is condensed, pick off the rod with the tip of a needle, and a cavity slot appears in the center.

2. At one end of the hole slot on both sides near the edge of the 2mm each punch a small hole (diameter of 2 - 3mm), in a hole hole in the healthy plant leaf sap, in the other hole to add the virus infected plant leaf sap.

3. Add the juice of the hole end connected to the negative pole, electrophoresis buffer is still 0.1 mol / LpH8.6 sodium barbital hydrochloric acid buffer, voltage 150V, electrophoresis 1h.

4. Add anti-HRP rabbit serum in the cavity tank, put it in a wet box, and react in a 37℃ warm box for about 3h or overnight at room temperature.

5. Dip and wash in saline for 3 - 4 days, change the water 3 times a day.

6. Apply clean wet filter paper and dry in 40℃ oven.

7. immersed in the substrate color development, from the shape of the precipitation arc, the site of the different, compare Jian, the precipitation line formed by the disease sap.

Common Problems

1. Positive reaction: a clear brownish-red line between the antigen and antibody wells.

2. Negative reaction: no brown-red line.

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