Antibodies in Practice: Publishing and Validating Antibodies

Publishing with Antibodies

Let’s return to the basic immunolabeling assumption that regardless of technique used, a positive signal infers that the specific antibody has bound to the specific antigen. As with any technique, it is good science to verify that your signal is indeed specific and reproducible. Reviewers of publications and grants are becoming increasingly critical of data analysis, and researchers are being challenged to think about the fundamental principles by which laboratory techniques work, and to be more careful about over-interpretation. The chart below should help bolster confidence in immunodata.


Actual reviewer comments Good practice
I am not convinced your antibody is specific
  • Use two or more different techniques to verify specificity; for example, WB can corroborate IHC data
  • Use two or more antibodies made against different immunogens or regions of the protein and measure co-localization
  • Test against relevant knockout samples
I am not convinced your antibody is not cross-reacting with related proteins
  • Use two or more antibodies made against unconserved epitopes of the same antigen to confirm results
Your Western blot shows more than one band or at the wrong size. How can you show specificity?
  • Cite published literature on cleavage products or glycosylation patterns
  • Consider running a denatured vs. native gel
  • Reprobe with a different antibody to same protein
Error bars are disturbingly large on your antibody-based data
  • Lock down your antibody protocol and then ensure you have enough antibody from the same lot number, so you don’t have to re-optimize each experiment because of lot-to-lot variability
How much of the signal is actually background?
  • Optimize protocol and reduce variability (see above).
  • Perform a peptide inhibition assay
  • Perform experiment without the primary antibody to establish background
  • Co-localize with direct fluorescent labeled primary
  • Use species preabsorbed secondary antibodies
Repeat your experiment with monoclonal antibodies for better data interpretation
  • Many monoclonals are available for targets recognized by polyclonals
  • Choose a polyclonal made from a short peptide thus minimizing clonality and epitope
  • Choose a polyclonal antibody validated in multiple applications to demonstrate specificity across sample matrices, epitope treatments and detection environment
Redo experiment using antibody with known epitope.
  • Many antibody sequences are published by researchers or commercial suppliers and can be requested
  • Sequenced epitopes are not necessary for verifying antibody specificity or experiment reproducibility
  • Publish antibody catalog number and company to aid in peer validation of your data

Validating Antibodies

Validation is a process whereby, through the use of specific laboratory procedures, the performance and characteristics of an analytical technique are deemed suitable for the intended use.

Usually, the first test for antibody specificity is Western blotting of a variety of cell line lysates with known levels of target expression. Here, both positive and negative control cells are used. The next step in the validation process is often immunohistochemistry (IHC) or immunofluorescence (IF) to titer the antibody on tissue samples.

During validation, it must be shown that not only are the antibodies specific and selective, but they can also provide reproducible results. Hence, reproducibility is the final step in the validation process.

Antibody validation is particularly important in immunohistochemistry applications. Analysis of IHC data can be challenging due to pre-analytical, analytical, and post-analytical factors that affect staining, particularly with free-floating or paraffin-embedded sections.

Standard Antibody Validation

Enhanced Antibody Validation