The Western Blot is performed by a process in three stages: gel electrophoresis (allowing separate proteins present in the sample), the transfer membrane and the addition of a specific antibody to detect the protein of interest.
Antibodies play a fundamental role in the successful resolution of this immunoassay, so the correct choice of antibodies for Western Blot becomes a critical point when performing them.
General Considerations For Choosing Western Blot Antibodies
1.- ANTIGEN-ANTIBODY INTERACTION
When carrying out a Western Blot in denaturing conditions, two important points should be kept in mind:
Antibodies that recognize conformational epitopes can lose this affinity if the protein is denatured.
Antibodies that recognize linear epitopes under denaturing conditions, may not detect them in the native structure of the protein, among others, for reasons of steric hindrance, for example.
2.- POLYCLONAL OR MONOCLONAL ANTIBODIES?
Or in other words, sensitivity or specificity? In general terms, polyclonal antibodies are more sensitive than monoclonal antibodies, which is especially relevant if you want to detect low protein in the sample.
The monoclonal antibodies however, due to its homogeneous nature, are much more specific so often result in lower background noise and minor problems of cross – reactivity to polyclonal.
In the case of secondary antibodies , the choice of Western Blot antibodies will also be conditioned by the detection method that we are going to use to visualize the presence of the protein of interest, since these must be marked differently depending on that the detection is carried out by colorimetry or by chemiluminescence.
With all this, we can conclude that the key to obtain a reliable and consistent result in Western Blot lies in the antigen-antibody interaction, and therefore, the choice of the antibodies for Western Blot should not be made only on the basis of the immunoassay, but also taking into account the specific aspects of our experiment such as the test conditions, the nature of the antigen or the detection method used.