The selection of the primary antibody is crucial for the success of a western blot experiment. The primary antibody is responsible for specifically binding to the protein of interest, which allows it to be visualized on the blot. There are several factors that must be considered when selecting a primary antibody:

  • Specificity: The primary antibody needs to be specific to the protein of interest. There should be no cross-reactivity with other proteins in the sample.
  • The primary antibody needs to be of high quality and properly validated. Choose an antibody that has been validated for western blotting. This means that the antibody has been shown to work in western blotting experiments and that it produces a strong signal
  • Affinity: The primary antibody should have a high affinity for the protein of interest. This means that it should bind to the protein tightly, even at low concentrations.
  • Concentration: The primary antibody should be used at the appropriate concentration; a proper titration protocol can be useful in determining the best concentration. Too much antibody can lead to background noise, while too little antibody may not produce a strong enough signal. Once a primary antibody has been selected, it is important to optimize the western blotting protocol to ensure that the antibody is being used at the optimal concentration and that the blot is being developed for the appropriate amount of time.
  • Host species: The primary antibody needs to be raised in a different species than the organism from which the sample was obtained. This is to avoid cross-reactivity with the endogenous immunoglobulins in the sample.
  • If possible, use an antibody that has been raised against a conserved region of the protein of interest. This will increase the likelihood that the antibody will work in different species.
  • Isotype: The primary antibody isotype should be compatible with the secondary antibody that will be used. It is also important to note that some secondary antibodies are raised against the Fab region of the primary antibody. The Fab region is the part of the antibody that binds to the antigen. Secondary antibodies that are raised against the Fab region must be specific for the isotype and subclass of the primary antibody.
  • Choose an antibody that has been validated for western blotting. This means that the antibody has been shown to work in western blotting experiments and that it produces a strong signal.
  • If possible, use an antibody that has been raised against a conserved region of the protein of interest. This will increase the likelihood that the antibody will work in different species.
  • Use a loading control antibody. This is an antibody that binds to a protein that is expressed in all cells. Loading control antibodies can be used to normalize the amount of protein loaded in each lane of the gel.
  • Employ automation when possible, to reduce the chance of human error. The BlotCyler is an ideal option to control all the blot processing step