The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/5000. Detects a band of approximately 268 kDa (predicted molecular weight: 292 kDa).
1/200. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Deubiquitinase involved both in the processing of ubiquitin precursors and of ubiquitinated proteins. May therefore play an important role regulatory role at the level of protein turnover by preventing degradation of proteins through the removal of conjugated ubiquitin. Essential component of TGF-beta/BMP signaling cascade. Regulates chromosome alignment and segregation in mitosis by regulating the localization of BIRC5/survivin to mitotic centromeres. Specifically hydrolyzes both 'Lys-29'- and 'Lys-33'-linked polyubiquitins chains. Specifically deubiquitinates monoubiquitinated SMAD4, opposing the activity of E3 ubiquitin-protein ligase TRIM33.
ab203471 was shown to specifically react with USP9x in wild-type HAP1 cells as signal was lost in USP9X knockout cells. Wild-type and USP9X knockout samples were subjected to SDS-PAGE. Ab203471 and ab184095 (Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control (Alexa Fluor® 680) loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.
IHC image of USP9x staining in a section of formalin-fixed paraffin-embedded human normal testis tissue sections*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab203471, 1/200 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.
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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
Western blot - Anti-USP9x antibody [EPR13809(B)] - N-terminal (HRP) (ab203471)
All lanes : Anti-USP9x antibody [EPR13809(B)] - N-terminal (HRP) (ab203471) at 1/5000 dilution
Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab203471 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.