This antibody gave a positive result in the following whole cell lysates: HeLa (Human epithelial carcinoma cell line)
Jurkat (Human T cell lymphoblast-like cell line)
A431 (Human epithelial carcinoma cell line)
HEK 293 (Human embryonic kidney cell line)
HepG2 (Human hepatocellular liver carcinoma cell line)
MCF-7 (Human breast adenocarcinoma cell line)
SHSY-5Y, U2OS (Human osteosarcoma cell line)
U2AF65 is necessary for the splicing of pre-mRNA. It binds to the polypyrimidine tract of introns early during spliceosome assembly. It is required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene (referenced from swissprot).
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
Primary antibody notesU2AF65 is necessary for the splicing of pre-mRNA. It binds to the polypyrimidine tract of introns early during spliceosome assembly. It is required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene (referenced from swissprot).
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
1/250. Detects a band of approximately 65 kDa (predicted molecular weight: 53 kDa).
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
FunctionNecessary for the splicing of pre-mRNA. Induces cardiac troponin-T (TNNT2) pre-mRNA exon inclusion in muscle. Regulates the TNNT2 exon 5 inclusion through competition with MBNL1. Binds preferentially to a single-stranded structure within the polypyrimidine tract of TNNT2 intron 4 during spliceosome assembly. Required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene. Represses the splicing of MAPT/Tau exon 10.
Sequence similaritiesBelongs to the splicing factor SR family. Contains 3 RRM (RNA recognition motif) domains.
Post-translational modificationsLysyl-hydroxylation at Lys-15 and Lys-276 affects the mRNA splicing activity of the protein, leading to regulate some, but not all, alternative splicing events.
ICC/IF image of ab37483 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab37483, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
IHC image of U2AF65 staining in human lymphoma FFPE section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab37483, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
References for Anti-U2AF65 antibody (ab37483)
This product has been referenced in:
Ammon T et al. The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells. J Mol Cell Biol6:312-23 (2014).
Read more (PubMed: 24872507) »
Heim A et al. Jumonji domain containing protein 6 (Jmjd6) modulates splicing and specifically interacts with arginine-serine-rich (RS) domains of SR- and SR-like proteins. Nucleic Acids Res42:7833-50 (2014).
Read more (PubMed: 24914048) »