This antibody gave a positive signal in the following human whole cell lysates: Hela, HEK293, Ramos.
This antibody gave a positive result in IHC in the following FFPE tissue: Human colon adenocarcinoma.
Shipped 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.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Detects a band of approximately 360 kDa (predicted molecular weight: 360 kDa).
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
E3 SUMO-protein ligase which facilitates SUMO1 and SUMO2 conjugation by UBE2I. Involved in transport factor (Ran-GTP, karyopherin)-mediated protein import via the F-G repeat-containing domain which acts as a docking site for substrates. Could also have isomerase or chaperone activity and may bind RNA or DNA. Component of the nuclear export pathway. Specific docking site for the nuclear export factor exportin-1.
Protein modification; protein sumoylation.
Involvement in disease
Defects in RANBP2 are the cause of susceptibility to encephalopathy acute necrotizing type 1 (ANE1) [MIM:608033]. A rapidly progressive encephalopathy manifesting in susceptibile individuals with seizures and coma. It can occur within days in otherwise healthy children after common viral infections such as influenza and parainfluenza, without evidence of viral infection of the brain or inflammatory cell infiltration. Brain T2-weighted magnetic resonance imaging reveals characteristic symmetric lesions present in the thalami, pons and brainstem.
IHC image of RanBP2 staining in Human colon adenocarcinoma 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 ab58385, 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 - RanBP2 antibody (ab58385)
All lanes : Anti-RanBP2 antibody [mAbcam58385] (ab58385) at 5 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate Lane 3 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lysates/proteins at 20 µg per lane.
Secondary Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 360 kDa Observed band size : 360 kDa Additional bands at : 220 kDa,230 kDa. We are unsure as to the identity of these extra bands.
Overlay histogram showing HeLa cells stained with ab58385 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab58385, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.