Learning the electrophoresis technique of separating proteins in the gel and the immunodiffusion technique of using the specific reaction of antigen and antibody, combined with the immunochemical method of analyzing and identifying the antigen or antibody.
(Source: Biochemistry and Molecular Biology Laboratory Guide, Shao Xueling et al, Wuhan University Press, 2003)
Operation method
immunoelectrophoresis
Principle
Immunoelectrophoresis is a method that combines the techniques of protein electrophoretic separation (agar electrophoresis) and immunological detection (bidirectional diffusion). This method has the advantages of high resolution, high sensitivity and short time on the basis of micro-volume, which makes it an ideal method for separating and identifying protein mixtures. It is ideal for the separation and identification of protein mixtures. It is used for the characterization of antigens and antibodies as well as for the determination of their purity. It is also valuable in clinical diagnosis. (Source: Laboratory Guide to Biochemistry and Molecular Biology, Shao Xueling et al, Wuhan University Press, 2003)
Materials and Instruments
Human IgG Rabbit Anti-Human IgG Move 1. Agar plate preparation For more product details, please visit Aladdin Scientific website.
Agar Sodium barbiturate Bovine serum albumin
Electrophoresis apparatus Horizontal electrophoresis bath Perforator Micro-sampler Toothpicks Slides Constant temperature bath Knife
Lay out the agar plate as shown below, then punch and scribe the groove as shown below, do not pick out the agar in the groove during electrophoresis (the two longitudinal parallel sides of the groove are not scribed first). 
2. Sampling
Make the upper wells of the agar plate filled with antigen solution and the lower wells filled with indicator protein.
3. Electrophoresis
As above, adjust the current intensity to 2~4 mA/cm. electrophoresis can be stopped when the indicator protein reaches the edge of the filter paper. Generally it takes 40～60 min.
4. Diffusion
After electrophoresis, pick out the agar gel from the groove in the middle of the agar plate with a toothpick and add the immune serum. Place the agar plate in a warm box (or petri dish) and leave it covered overnight. The following day you can observe the precipitation lines that appear within the gel. In general each precipitation line represents an antigenic component. Based on the number and location of the precipitates, the purity of the antigen and the type of antigenic protein can be determined. Microimmunoelectrophoresis is commonly used to analyze the serum composition of samples.
5. Observation
The graph of immunoelectrophoresis can be observed directly as antigen-antibody precipitation lines or after staining with 0.1% amino black. Photographs can be taken if needed for preservation.