All lanes : Anti-RAP1GAP antibody [Y134] (ab32373) at 1/10000 dilution (purified)

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate

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: 95 kDa
Observed band size: 95 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Anti-RAP1GAP antibody [Y134] (ab32373) at 1/10000 dilution (purified) + Jurkat cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 95 kDa
Observed band size: 95 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Anti-RAP1GAP antibody [Y134] (ab32373) at 1/10000 dilution (purified) + SH-SY5Y cell lysate at 20 µg

Secondary
Anti-rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 95 kDa
Observed band size: 95 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Anti-RAP1GAP antibody [Y134] (ab32373) at 1/50000 dilution (unpurified) + Jurkat cell lysate

Predicted band size: 95 kDa
Observed band size: 95 kDa

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab32373 at a working dilution of 1/1000. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin-embedded human breast cancer tissue using unpurified ab32373 at 1/100 dilution.

Immunofluorescence staining of HeLa cells with purified ab32373 at a working dilution of 1/250, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4 % PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab32373 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

ICC/IF image of unpurified ab32373 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32373, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight^® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h.Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Overlay histogram showing Jurkat cells fixed in 4% PFA and stained with purified ab32373 at a dilution of 1/30 (red line). The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Overlay histogram showing SH-SY5Y cells stained with unpurified ab32373 (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 (ab32373, 1/100) 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. Acquisition of >5,000 events was performed.

ab32373 (purified) at 1/20 immunoprecipitating RAP1GAP in 10 μg C6 cell lysate (Lanes 1 and 2, observed at 82-95 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730). For western blotting, HRP Veriblot for IP Detection Reagent (ab131366) was used for detection (1/1000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST