Western FAQ Analysis (1)

Choosing the right buffer is important to maintain protein stability at a certain pH and to ensure repeatability of the experiment. The buffering capacity of PBS is stronger than that of TBS, and the buffering capacity of TBS is lower than pH 7.0. Generally, a suitable buffer is selected according to the purpose of the experiment, such as anion exchange chromatography in protein purification, TBS is preferred for the cation buffer, and PBS is preferred for the anion buffer during cation exchange chromatography. Both PBS and TBS can be used in Western blot, and the appropriate concentration can be selected as needed.

Not necessarily, because some antibodies recognize linear epitopes, some recognize conformational epitopes, and antibodies that recognize conformational epitopes cannot be used for Western blots because antibodies recognize fully degenerated protein antigens in Western blots. The conformational epitope of the protein antigen is destroyed when it is denatured.

It is best not to add two primary antibodies at the same time, because if there are non-specific signals in the results, it is not clear. Western is used to detect the target protein and internal control on the same membrane. It is also the primary antibody, secondary antibody, ECL, color, tablet, and wash of the target protein. After rinsing, add the primary antibody, secondary antibody, and ECL. Color, tablet, and wash.

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.

The concentration of the primary antibody or the secondary antibody is too high, the non-specific reaction of the polyclonal antibody itself, the specificity of the antibody is not strong, the target protein changes after treatment, and the bands of the internal reference are substantially uniform, so that it is convincing. It is indicated that the treatment factor causes the change of the target protein instead of the error of loading or artificially causing the change of the concentration of the target strip. In the strict sense, the internal reference is necessary.