All lanes : Anti-RanGAP1 antibody [EPR3295] (ab92360) at 1/1000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates
Lane 3 : Mouse brain lysates
Lane 4 : Rat brain lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 64 kDa
Observed band size: 70,90 kDa why is the actual band size different from the predicted?

The doublets detected are consistent with what have been described in literature PMID: 24988324 and PMID: 21646468

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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue sections labeling RanGAP1 with purified ab92360 at 1/500 dilution (0.22 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling RanGAP1 with purified ab92360 at 1/100 dilution (1.1 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 µg/ml) dilution. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling RanGAP1 with purified ab92360 at 1/20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

ab92360 (purified) at 1/20 dilution (0.5ug) immunoprecipitating RanGAP1 in HeLa whole cell lysates.
Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates 10ug
Lane 2 (+): ab92360 & HeLa whole cell lysates
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab92360 in HeLa whole cell lysates
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue sections labeling RanGAP1 with purified ab92360 at 1/500 dilution (0.22 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue sections labeling RanGAP1 with purified ab92360 at 1/500 dilution (0.22 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

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.

b92360 at 1/100 dilution staining RanGAP1 in paraffin-embedded (1) Human breast carcinoma tissue and (2) Human testis tissue by immunohistochemistry.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

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.

See Abreview

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.