Novel Mutation Specific Histone Antibodies generated by Cambridge Research Biochemicals

In a collaboration with Professor Richard Grundy and Dr Farhana Haque at the Children’s Brain Tumour Research Centre (CBTRC), Layfield and co-workers employed novel mutation specific custom polyclonal antibodies generated by Cambridge Research Biochemicals to discriminate missense (G34R and G34V) mutant histone H3 (H3.3) proteins from their wild type counterparts.

The antibodies
generated were shown to detect both exogenous and endogenous H3.3 G34R and H3.3 G34V mutant proteins by Western Blot and immunocytochemistry. The research focus of the group, based at the University of Nottingham, includes a range of different brain tumours.

Peptide sequences were carefully designed to ensure specificity to the single mutated residues along with the incorporation of highly refined affinity chromatography processes to ensure effective removal of wild-type specific antibodies. ELISA analysis of the anti-sera generated throughout the course of the immunisation programmes demonstrated selectivity towards the mutant sequence when compared to the wild type analogue.

Immunofluorescence: The figure shown right indicates antibody staining of patient derived cell lines with histone mutations, detected by Immunofluorescence microscopy. The H3-G34R and H3-G34V antibodies were generated by Cambridge Research Biochemicals.

Western Blot: The Western blot below shows selective detection of purified recombinant GST-mutant specific histone proteins from the wild type sequences with antibodies, H3-G34R and H3-G34V, generated by Cambridge Research Biochemicals.

To read more about these antibodies please refer to the following citation:

Haque F, Varlet P, Puntonet J, Storer L, Bountali A, Rahman R, Grill J, Carcaboso AM, Jones C, Layfield R, Jones C, Layfield R and Grundy RG. (2017) Evaluation of a novel antibody to define histone 3.3 G34R mutant brain tumours. Acta Neuropathologica Communications. 5; 45

Cambridge Research Biochemicals are world leaders in peptide synthesis (antigen peptides and other variants/ modifications) and antibody generation, with over 38 years of
industry expertise. Mutant specific anti-peptide antibodies are highly sought-after research tools for gaining knowledge and insight into target specific mutations.

These highly specific antibodies will soon be available via CRB’s partner brand DISCOVERY antibodies – click here to visit the website to browse in the meantime. If you would like to express an interest in this antibody, please email the DISCOVERY antibodies Team.

TESTIMONIAL

Professor Rob Layfield, University of Nottingham

“We were delighted when Cambridge Research Biochemicals were able to help us generate and purify the mutant-specific antibodies – this was a risky project and downstream work was wholly dependent on this being successful.

What really surprised us was that the production of such reagents is far from commonplace. For example hundreds of different phospho-specific antibodies have been developed, but only a handful of mutant-specific ones.

The purified antibodies from Cambridge Research Biochemicals exceeded our expectations and have already provided new insights into tumour biology.”