In western blotting the primary antibody is the reagent used to detect the protein of interest. The antibody is added after membrane blocking, the primary antibody binds specifically to the protein of interest. The membrane is incubated with the primary antibody at room temperature or preferably overnight incubation at 4°C. Following incubation with the primary antibody the membrane is washed to remove any excess and unbound antibody. What are the considerations for selecting the primary antibody? This depends on a number of factors. The immunogens used to produce the primary antibody will directly affect whether they are suitable candidates. A poor immunogen will lead to unreliable and potentially irreproducible results. A peptide based immunogen can be useful when an antibody needs to differentiate between two proteins having a highly conserved amino acid sequence. Native proteins that are used as immunogens can be problematic; some antibodies raised using native protein will only recognize the epitopes on the surface of a protein in its native, oxidized conformation. This is especially true of monoclonal antibodies.
The good news is there are a number of companies that manufacture quality primary antibodies that fit specific criteria based on the protein of interest e.g. phosphorylated proteins. In order to detect the protein of interest, the concentration of primary antibody is very important. A sub-optimal concentration could result in a number of problems which include: poor signal, nonspecific bands and background noise. There are a number of factors that determine the optimal concentration of antibody: specificity of the antibody, antigen concentration, antibody affinity, experimental conditions (# of washes, buffer composition, running at RT or 4 degrees, etc.)
The Western blot process is a time-consuming delicate process; a small imbalance at any level of the procedure can skew the results of the entire process. Performing the Western blot using a manual procedure even with a highly trained technician can also be problematic. The ability to reproduce exact time points for blocking and washing can only be realistically done implementing an automated procedure. A number of researchers using the BlotCycler Touch or Blotcycler Mini achieve better sensitivity and consistent reproducible results; researchers can put there stop watches away and start using their time more efficiently.
