1/100 - 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
1/100 - 1/250.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
GTPase activator for the nuclear Ras-related regulatory protein Ran, converting it to the putatively inactive GDP-bound state.
Highly expressed in brain, thymus and testis.
Belongs to the RNA1 family. Contains 6 LRR (leucine-rich) repeats.
Phosphorylated occurs before nuclear envelope breakdown and continues throughout mitosis. Phosphorylated by the M-phase kinase cyclin B/Cdk1, in vitro. Differential timimg of dephosphorylation occurs during phases of mitosis. The phosphorylated form remains associated with RANBP2/NUP358 and the SUMO E2-conjugating enzyme, UBC9, on nuclear pore complex (NPC) diassembly and during mitosis. Sumoylated with SUMO1. Sumoylation is necessary for targeting to the nuclear envelope (NE), and for association with mitotic spindles and kinetochores during mitosis. Also required for interaction with RANBP2 and is mediated by UBC9.
Cytoplasm. Nucleus membrane. Chromosome, centromere, kinetochore. Cytoplasm, cytoskeleton, spindle pole. Cytoplasmic during interphase. Targeted to the nuclear rim after sumoylation. During mitosis, associates with mitotic spindles. Association with kinetochores appears soon after nuclear envelope breakdown and persists until late anaphase. Mitotic location also requires sumoylation.
Immunofluorescence staining of MCF7 cells with purified ab92360 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was an Alexa Fluor® 488 conjugated goat anti-rabbit (ab150077), used at a dilution of 1/1000. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative control is shown in bottom right hand panel - for the negative control, PBS was used instead of the primary antibody.
Western blot - RanGAP1 antibody [EPR3295] (ab92360)
All lanes : Anti-RanGAP1 antibody [EPR3295] (ab92360) at 1/1000 dilution
Lane 1 : HeLa cell lysate Lane 2 : MCF-7 cell lysate Lane 3 : SH-SY5Y cell lysate Lane 4 : A549 cell lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : HRP labelled goat anti-rabbit antibody at 1/2000 dilution
Predicted band size: 64 kDa Observed band size: 64 kDa Additional bands at: 90 kDa. We are unsure as to the identity of these extra bands.
b92360 at 1/100 dilution staining RanGAP1 in paraffin-embedded (1) Human breast carcinoma tissue and (2) Human testis tissue by immunohistochemistry.
Immunocytochemistry/ Immunofluorescence - Anti-RanGAP1 antibody [EPR3295] (ab92360)This image is courtesy of an anonymous Abreview
ab92360 staining RanGAP1 in mouse hepatocyte cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized, and blocked with 2% BSA for 2 hours at 22°C. Samples were incubated with primary antibody (1/100 in blocking buffer) for 18 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/10000) was used as the secondary antibody.
Overlay histogram showing Jurkat cells stained with ab92360 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab92360, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
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