Analysis of common problems in Western (2)

In the case of Western, in theory, the specificity of the monoclonal antibody is better than that of the polyclonal antibody, but the type of the monoclonal antibody is relatively small, and the general price is high, so generally the resistance is sufficient. Antibodies for Western and antibodies for ELISA, generally used for Western antibodies to recognize amino acid sequence specificity, while antibodies for ELISA depend on antigen type, some identify amino acid sequence specificity, and some identify Conformational specificity, so it is generally determined according to the instructions. There are two main considerations for immunoblotting antibody selection. Whether the selected antibody recognizes the denatured protein transferred to the membrane after gel electrophoresis, and the other is whether the selected antibody causes a cross-reactive band.

It can be equal, but if the paper or film is larger than the gel, it is easier to form a short circuit. The upper and lower layers of filter paper should not be in contact with each other because they are too large. This will short-circuit and the current will not pass through the glue and filter paper. Look at the voltage before and during the transfer. The normal semi-dry rotation is slowly getting higher. At the end, it is generally 1.5-3 times the beginning is normal.

Anionic dyes are more commonly used, especially amino black, which has fast decolorization and low background detection limit of 1.5ug. Although Coomassie Brilliant Blue has the same sensitivity as amino black, it has slow decolorization and high background. Ponceau S and Fast Green are easily removed from the protein after detection for subsequent amino acid analysis.
Colloidal gold, high sensitivity, detection range can reach pg level, but dyeing is stable.
The biotin sensitivity is between 1.2 and can be used on any membrane.

It can be considered that 20% methanol is added to the transfer buffer because methanol can reduce the protein elution efficiency, but can increase the binding ability of protein and NC membrane, methanol can prevent gel deformation, methanol can prolong the transfer time for high molecular weight protein; The final concentration of 0.1% SDS was added to the buffer to increase the transfer efficiency; use a high quality transfer film, or use a small pore size NC membrane, low concentration gel to increase the transfer voltage/current and increase the transfer time.

DAB coloration can form a gray-brown terminal product in the action of horseradish peroxidase. The product is insoluble in alcohol and other organic solvents. The oxidation of DAB can also cause polymerization, resulting in an increase in reaction with osmium tetroxide. Its dyeing strength and electron density.