The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration. PubMed: 27252533
Use a concentration of 1 µg/ml.
Use at an assay dependent concentration. PubMed: 21725067
1/250. Detects a band of approximately 65 kDa (predicted molecular weight: 53 kDa).
Necessary 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.
Belongs to the splicing factor SR family. Contains 3 RRM (RNA recognition motif) domains.
Lysyl-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 ab37530 stained human HeLa cells. The cells were PFA fixed (10 min) and incubated with the antibody (ab37530, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used to quench autofluorescence.5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
IHC image of ab37530 staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue 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 ab37530, 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - Anti-U2AF65 antibody (ab37530)
All lanes : Anti-U2AF65 antibody (ab37530) at 1/250 dilution
Lane 1 : Marker Lane 2 : Zebrafish brain homogenate at 20 µg Lane 3 : Zebrafish heart homogenate at 10 µg Lane 4 : Zebrafish liver homogenate at 10 µg Lane 5 : Zebrafish skeletal muscle homogenate at 10 µg Lane 6 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
Secondary All lanes : Goat polyclonal to Rabbit IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 53 kDa Observed band size: 53 kDa
U2AF65 was immunoprecipitated using 0.5mg SHSY5Y whole cell extract, 5µg of Rabbit polyclonal to U2AF65 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, SHSY5Y whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab37530. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 65kDa: U2AF65.
Hou W et al. Two Polypyrimidine Tracts in Intron 4 of the Major Immediate Early Gene Are Critical for Gene Expression Switching from IE1 to IE2 and for Replication of Human Cytomegalovirus. J Virol90:7339-49 (2016).
Read more (PubMed: 27252533) »
Tamayo AG et al. Histone monoubiquitination by Clock-Bmal1 complex marks Per1 and Per2 genes for circadian feedback. Nat Struct Mol Biol22:759-66 (2015).
Read more (PubMed: 26323038) »