Christian Frezza et al. at the MRC Cancer Unit, Cambridge chose to enlist the assistance of Cambridge Research Biochemicals (CRB) to raise custom polyclonal antibodies against protein succination, a post-translational modification formed by a reaction between the tricarboxylic acid cycle intermediate fumarate with protein cysteines to form S-(2-succino)cysteine (2SC).
“We contacted CRB for the generation of the 2SC antibody, a key tool for our research programme. We knew this was a challenge, but CRB reassured us that they would do their best to help”
The antigen was carefully designed to ensure specificity to the cysteine modification, along with exacting screening methods highly refined affinity chromatography processes to ensure effective purification of specific antibodies.
2SC is considered as a biomarker for mitochondrial stress in obesity, insulin resistance and diabetes, with succination contributing to reduced levels of plasma adiponectin in type 2 diabetic mice (Nagai et al., 2007). Furthermore, Succination at critical cysteine residues can result in the inactivation of enzymatic activity or protein function in many biological processes and the succination of glutathione has been shown to increase oxidative stress and cellular senescence. Therefore, the elucidation of the succinated proteome will provide an insight into the role of succination in regulatory biology and determine its effect on cellular dysfunction. Read more about the role of Succination here.
Chrstian Frezza comments, ‘This antibody is an essential tool for our research programme, which is focused on understanding the consequences of the loss of the tumour suppressor Fumarate Hydratase. We will use this antibody as surrogate for the accumulation of fumarate, the substrate of Fumarate Hydratase, strikingly accumulated in Fumarate Hydratase deficient tumours. Furthermore, this antibody will be essential to identify new targets of succination driven by fumarate accumulation, not only in cancer but also in diabetes, where succination was originally observed.”
“CRB have been extremely helpful and approachable both during the development and after the production of the antibodies. I would recommend them highly.”
“After an initial discussion about the procedure to follow for the immunization and antigen generation, CRB quickly delivered the first batch of antibody. We had a very productive series of iterations for the optimisation of the antibody, and we had our first working batch within few months. I am very impressed with the efficiency of CRB and their attitude towards this project.”
CRB are experts at generating custom made antisera against a wide and complex range of targets to include post-translational modifications, peptides, proteins and drug antibodies and have refined their in-house methodology over 39 years of business. Consultation on peptide design and perfected purification techniques help to encourage project success and the project can be tailored to suit the customers’ requirements.
CRB have an excellent publication record which can be viewed here.