Antibody validation is the process of ensuring that an antibody specifically recognizes and binds to its intended target molecule, with minimal non-specific binding. It’s critical for guaranteeing the reliability and reproducibility of research results, especially in downstream applications like immunohistochemistry, western blotting, and ELISA.

There are a number of validation parameters to consider, and the specific standards vary depending on the application and the target molecule.

Specificity:

  • Western blotting: A specific antibody should produce a single band at the expected molecular weight of the target protein, with minimal background staining.
  • Immunohistochemistry (IHC): Staining should be localized to the expected cellular or tissue compartments where the target protein is known to be expressed, with minimal staining in control tissues or cells lacking the target protein.
  • ELISA: A specific antibody should show a dose-dependent increase in signal with increasing concentrations of the target protein, with minimal non-specific binding to other components in the sample.

Sensitivity:

  • The antibody should be able to detect the target protein at low concentrations, relevant to your experimental conditions.

Reproducibility:

  • The antibody should produce consistent results between different experiments. By including automation in the Blot processing steps reproducibility is much less of a problem. Both the BlotCycler Touch and Mini automate all blot processing steps: blocking, primary Ab, secondary Ab, and all washing steps. The automated process is conducted at 4 degrees C which is important for both primary Ab and washing

Negative controls:

  • Always include negative controls in your experiments to assess non-specific binding. These controls may include:
    • Tissue or cells lacking the target protein
    • Blocking peptides that compete with the antibody for binding to the target protein
    • Isotype control antibodies of the same species but raised against an unrelated antigen

Positive controls:

  • Include positive controls to ensure that your experimental system is working properly. These controls may include:
    • Purified target protein
    • Cells or tissues known to express the target protein

Automated western blot processing and DNA/RNA electrophoresis.