Affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific phosphopeptide. The antibody against non-phosphopeptide was removed by chromatography using non-phosphopeptide corresponding to the phosphorylation site.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/500 - 1/1000. Detects a band of approximately 58 kDa (predicted molecular weight: 58 kDa).
Involved in intracellular signal transduction mediated by cytokines and growth factors. Upon IL-2 and GM-CSL stimulation, it plays a role in signaling leading to DNA synthesis and MYC induction. May also play a role in T-cell development. Involved in down-regulation of receptor tyrosine kinase via multivesicular body (MVBs) when complexed with HGS (ESCRT-0 complex). The ESCRT-0 complex binds ubiquitin and acts as sorting machinery that recognizes ubiquitinated receptors and transfers them to further sequential lysosomal sorting/trafficking processes.
The VHS and UIM domains mediate the interaction with ubiquitinated proteins. The SH3 domain mediates the interaction with USP8. Contains one Pro-Xaa-Val-Xaa-Leu (PxVxL) motif, which is required for interaction with chromoshadow domains. This motif requires additional residues -7, -6, +4 and +5 of the central Val which contact the chromoshadow domain.
Phosphorylated in response to IL-2, GM-CSF, EGF and PDGF.
IHC image of STAM2 (phospho Y192) staining in human lung formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab63569, 1µg/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 - STAM2 (phospho Y192) antibody (ab63569)
All lanes : Anti-STAM2 (phospho Y192) antibody (ab63569) at 1/500 dilution
Lane 1 : Extracts from NIH/3T3 cells (5-30µg),
treated with EGF (200ng/ml, 30mins), minus immunising peptide. Lane 2 : Extracts from NIH/3T3 cells (5-30µg),
treated with EGF (200ng/ml, 30mins), plus immunising peptide (5-10µg).
Predicted band size : 58 kDa Observed band size : 58 kDa