Chapter 3: Immunodetection

After blotting, the target protein will be detected using appropriately matched and labeled antibodies . The typical immunodetection stage involves a few basic steps:

  • Blocking - The blot containing the transferred protein bands is incubated with a protein or detergent solution which covers the entire surface so that antibodies do not bind non-specifically to the membrane.
  • Antibody incubation - Labeled antibody binds to the target protein band present on the blot in a one-step or two-step procedure. 
  • Detection with substrate - The label attached to the antibody, usually an enzyme such as HrP (Horseradish Peroxidase), is detected using a substrate which produces a visible signal corresponding to the position of the target protein.

Figure 10: Immunodetection Overview. The Western blot is blocked, incubated with antibodies, and treated with substrate to make the target protein visible . Wash steps are carried out between incubations to remove excess unbound material and to minimize non-specific signal on the immunoblot.

 

 


Blotting Immunodetection: Blocking and Antibody Incubation