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.
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.
Antibodies are essential reagents that support all levels of scientific research. Used in a multitude of applications to identify, quantify, and isolate specific target biomolecules, they have recently become the focus of intense scrutiny for their contribution to the ongoing reproducibility crisis.
Have you ever wondered about the minds behind our antibodies? We talk a lot about validation, specificity, sensitivity, and reproducibility. All of that is very important, but that doesn't tell you much about who developed it.
When you’re shopping for antibodies, there are so many factors to consider. For example, will it work in my cell or tissue model? Has it been tested in the application I want to use? Sometimes it’s a struggle to find what you need because your options are limited, but in other instances there may be several reagents that seem like they could work in your experiment.
Researchers who run a lot of chromatin immunoprecipitation "ChIP" assays – maybe even your advisor – might subscribe to the idea that polyclonal antibodies perform better than monoclonal antibodies. But is that always actually true?
It’s worth your time to understand the differences between the two in terms of antigen recognition and specificity, and dispel some myths.