Biological research is about expanding knowledge, so it's not uncommon for scientists to become interested in studying proteins for which antibodies have not yet been developed. How then, can scientists pursue mechanistic studies of these novel or less-characterized proteins? Epitope tagging is a powerful tool for these protein studies and is used in myriad experimental applications. However, care must be taken not only in choice of epitope tag, but in selection and validation of antibodies.
Topics: Antibody Performance
During this time of crisis, healthcare providers and researchers around the world are racing to develop SARS-CoV-2 testing methods. Testing strategies include PCR to diagnose active infection (by testing for the presence of viral genetic material) and the use of antibody test kits on blood (also known as serological tests), in order to track exposure and possible immunity in the population (1,2,3). With these diagnostic tools, the scientific community aims to get a better understanding of the scale and trajectory of the SARS-CoV-2 pandemic.
If one were asked to describe a Cell Signaling Technology (CST) antibody sampler kit, we would totally understand an answer like, “it’s an assortment of antibodies.” Honestly, on its face, that answer isn’t exactly wrong. It just makes the process seem a little random. Especially considering the reality:
Depending on the antigenic target being studied and the application being used, it may be advisable to employ complementary strategies during antibody validation. These approaches can provide vital information regarding antibody specificity or functionality and can be carefully tailored to the biological nature of the target as well as to the exacting requirements of the downstream assay.
For antigenic targets where expression of the protein is very low or unknown, the use of recombinant proteins or exogenous expression in a surrogate cell line may be necessary for antibody validation. Although endogenous systems are preferred for their closer representation of in vivo conditions, heterologous strategy offers several advantages.
A multiple antibody strategy is a powerful approach to antibody validation. One of the most common methods to achieve this is to immunoprecipitate (IP) the target with one antibody and subsequently detect it by western blotting with another antibody against the same target. This provides confidence that both antibodies are binding the correct biomolecule.
We’re honored to win two CiteAb awards in 2020: Antibody Supplier of the Decade and Antibody Supplier Succeeding in Alzheimer’s Research. CST was also highly commended as an ELISA Kit Supplier to Watch in 2020. It’s always nice to be recognized for your work. In this case, it’s especially sweet because both efforts are near and dear to our hearts.
Topics: Antibody Performance
An orthogonal strategy for antibody validation involves cross-referencing antibody-based results with data obtained using non-antibody-based methods. This approach is critical to verify existing antibody validation data and to identify any effects or artifacts that are directly related to the antibody in question. Providing an additional level of detail to support results generated by the other strategies outlined within this handbook, orthogonal validation often utilizes data which are available in the public domain.
While a binary approach is one of the more desirable ways to evaluate antibody specificity, binary models are not always readily available and can be time consuming or expensive to produce for the sole purpose of validating an antibody. Moreover, to assess the sensitivity of an antibody in the application and protocol being used, a complementary hallmark is required.
A binary approach is one of the best ways to evaluate antibody specificity. By testing an antibody in biologically relevant positive and negative expression systems, it is possible to confirm that it recognizes the target antigen in its native environment without cross-reacting with other biomolecules present in the sample.