YCharOS (Antibody Characterization through Open Science)

Can YCharOS recommend/score antibodies based on their performance in each tested application?

After presenting the YCharOS initiative on several occasions we have found that, for the most part, scientists interested in our reports have the expertise to interpret the antibody characterization data. Moreover, as the antibodies are tested under one specific set of conditions, the scoring/recommendation would be valid only under the precise experimental setup and cell line used. That said, YCharOS reports serve as an invaluable guide pointing scientists to select the appropriate antibodies for their experimental needs.

What determines a successful antibody in Western Blot?

A successful antibody immunodetects the target protein, and the signal observed in the wild-type (WT) lysate is lost in the knockout (KO) lysates. Ideally, the antibody also does not recognize any other protein under the conditions tested.

What determines a successful antibody in Immunoprecipitation?

Under the conditions used, a successful antibody immunocaptures the target protein to at least 10% of the starting material, which leads to an observable depletion of the target protein from the starting material.

What determines a successful antibody in Immunofluorescence?

A successful antibody immunolocalizes the target protein by generating a signal in WT cells that is at least 1.5-fold over the signal in KO cells. Signal provided by such antibody can be easily distinguished from background noise.

Why doesn’t YCharOS test the antibodies against a diseased or mutated version of target protein in human cell lines?

Although this would be a great follow-up study, YCharOS is currently focused on characterizing antibodies for every human protein, making this degree of complexity outside the scope of our study. As for now, our aim is to assist researchers in selecting high-quality antibodies for their respective areas of expertise.

Why aren’t the full blots shown from the immunoprecipitation results?

To ensure a standardized format, for all antibodies tested by immunoprecipitation, we first perform Western Blot with an antibody that has been previously KO-validated. Firstly, due to the lack of antibody availability, we tend to cut the membrane around the protein of interest. Secondly, when possible, we cut around the area of the membranes with IgG antibodies to avoid cross-reactivity during the Western Blot process of the immunoprecipitation protocol. Both scenarios account for the lack of full immunoprecipitation blots present in our results.

Why don’t you share information on antibody epitope regions?

YCharOS is a third-party company that prefers to remain unbiased to the performance of antibodies. We believe it is up to the experts to assess the relevance of epitope regions according to their research program.

Why aren’t the antibodies tested for Immunohistochemistry (IHC)?

Due to the lack of available human KO tissues for every human protein target, it would be difficult to characterize the commercial antibodies in IHC using our standardized procedure which evaluates antibodies by comparing their performance in WT and KO cell lines.         

Why do you use knockout cell lines as a control?

To detect whether a commercial antibody is potent and selective for its protein target, we have developed a comprehensive and standardized protocol that compares antibodies side-by-side in WT and KO cell lines. The KO cell lines are prepared using CRISPR/Cas9 technology, which uses gene editing to remove the protein target in the selected cell line. Thus, the antibody is expected to produce a signal in the WT cell lines but not in the KO cell lines as it does not express the target antigen. This method of comparing the parental and KO cell lines can help identify whether the antibody is accurately recognizing the intended protein target. In the case that the protein of interest is essential for cell viability, a knockdown approach is used and the same experimental workflow is followed.