VISUALIZING CATESTATIN AND CHROMOGRANIN A : An Antibody Validation

Abstract: Chromogranin A (CgA) and its cleavage product Catestatin (CST) are two very interesting proteins, especially due to their possible involvement in the development of type 1 diabetes. There are currently only a few primary antibodies against CgA and CST, and the ones that do exist are often only recommended for use on one method. Recently though, two new multifunctional antibodies were released, which are said to function on multiple methods. The aim of this project was to validate these two antibodies, one being the Chromogranin A Polyclonal Antibody (PA5-35071) from Thermo Fischer Scientific and the other being the Catestatin Polyclonal Antibody (289-MM-0288) from MediMabs. The methods of validation for these antibodies were western blot, flow cytometry, and immunofluorescent staining of frozen tissue. All study material were from mice and the tissues were chosen due to their established expression of CgA and CST. The results show that the antibodies are highly appropriate for use on flow cytometry and immunofluorescent staining of frozen tissue. The most appropriate concentration for the CgA-antibody was deemed as 1:25 for both flow cytometry and immunofluorescent staining. The most appropriate concentrations for the CST-antibody were deemed as 1:800 for flow cytometry and 1:1000 for immunofluorescent staining. However, the antibodies could not be validated for use on western blot. Further investigations are needed to determine if the antibodies are functional in western blot analysis or not. The validation performed here is an important first step in studying the involvement of these proteins in type 1 diabetes onset.