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26 Oct 2021

A high quality antibody against the T2A peptide

Andrew McKenzie and his team from the MRC Laboratory of Molecular Biology (LMB) enlisted the expertise of the team at Cambridge Research Biochemicals (CRB) to produce a highly specific antibody to the 2A peptide sequence. 2A was discovered in the foot-and-mouth disease virus (FMDV, a picornavirus) and is used in cloning vectors.

The team at MRC LMB required a high affinity, highly specific antibody for use in ChIP-seq experiments and chose to raise an antibody against the 2A self cleaving peptide. The small 2A peptide sequences, when cloned between genes, allows for efficient, stoichiometric production of discrete protein products within a single vector through a novel “cleavage” event within the 2A peptide sequence. 2A acts through ribosomal skipping to allow for the encoding of polyproteins which can dissociate into individual proteins upon translation. 2A peptide-linked polycistronic vectors can be used to express multiple proteins from a single open reading frame (ORF).

As part of their investigations into the regulation of lineage commitment in type 2 innate lymphoid cells (ILC2) Andrew McKenzie and his team wanted to identify targets of the RORα transcription factor. However, as commercial RORα antibodies were not of sufficient quality for ChIP-seq this led them to their decision to use the 2A peptide sequence as the antigen instead. The 2A sequence remains associated with RORα following expression in transgenic reporter mice (RoraTeal/Teal). With their new custom antibody from CRB they were able to successfully identify transcription targets of RORα.

Andrew McKenzie comments “CRB provided an excellent service, helping to optimise the peptide design to produce extremely useful antibodies for our detection of T2A-tagged proteins”.

Western blot analysis from Human embryonic kidney (HEK) cells transiently transfected with overexpressing constructs: RORα-FLAG-T2A (62 kD), RORα-FLAG (58 kD) or GATA-3-HA-T2A (48 kD), immunoprecipitated with anti-T2A or anti-FLAG antibody and detected
using anti-FLAG or anti-T2A antibody, respectively. Image courtesy of A. McKenzie.

Anti-T2A antibody is an excellent tool for researchers using 2A peptide based expression systems and can be used in a wide range of settings and is of the highest quality being suitable for ChIP-seq assays.

If you are interested in furthering your own research discoveries with a custom antibody against your protein of interest from CRB then please contact us here.