5 Pillars of Antibody Validation

At Novus, we understand how important it is to provide high quality, well validated research reagents. We recognize the need to enhance antibody validation for our customers. In addition to our current validation procedures, Novus will now implement several new methods for antibody validation, in accordance with recommendations instigated by the International Working Group for Antibody Validation (IWGAV)1.

We are committed to improving our validation standards. You can track Novus’ progress by looking on our website and product information sheets for the certification icons below:

genetic strategic validation Genetic Strategy Validation- Expression of the target protein is compared before and after knockout or knockdown using CRISPR/CAS9 or siRNA/shRNA. If protein expression following knockout or knockdown is substantially reduced, then antibody specificity is ensured.
orthogonal validation Orthogonal Validation-The target protein is examined with an antibody independent strategy and compared with results from an antibody-dependent strategy. A correlation between these two strategies indicates specificity between the antibody and its target protein. Examples of antibody independent techniques may include in situ hybridization, quantitative PCR, RNA-seq or mass spectrometry.
independent antibody validation Independent Antibody Validation- The data generated using several antibodies (ideally targeting different epitopes) in the same protein is compared (e.g. molecular weight and cellular localization).  Consistent results imply antibody selectivity to the target protein.
expression of tagged proteins Expression of Tagged Proteins Validation – A tagged protein is used as a standard for comparison in Western blotting and/or immunocytochemistry (ICC). For example, if the distribution of the tagged protein overlaps with the immunofluorescence signal, then antibody specificity is confirmed.
biological strategies validation Biological Strategies Validation- These strategies use defined biological or chemical modulation of protein expression to demonstrate antibody specificity to the target protein. The data is compared across multiple cell lines including positive and negative expressing cells, and multiple species, if applicable.

Learn more about Novus' Reproducibility Initiative


For more information regarding these validation techniques and background to the antibody validation crisis please see-  

Uhlen M, Bandrowski A, Carr S, Edwards A, Ellenberg J, et al. (2016) A proposal for validation of antibodies. Nature Methods 13(10):823-7