Anti-RAP1GAP antibody [Y134]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAP1GAP antibody [Y134] (AB32373)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RAP1GAP antibody [Y134] (AB32373)

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RAP1GAP antibody [Y134] (AB32373)

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 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5000 events was performed.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RAP1GAP antibody [Y134] (AB32373)

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

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-RAP1GAP antibody [Y134] (AB32373)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RAP1GAP antibody [Y134] (AB32373)

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).

• IP

Unknown

Immunoprecipitation - Anti-RAP1GAP antibody [Y134] (AB32373)

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

All lanes:

Immunoprecipitation - Anti-RAP1GAP antibody [Y134] (ab32373)

Predicted band size: 73 kDa

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• WB

Lab

Western blot - Anti-RAP1GAP antibody [Y134] (AB32373)

Western blot : Anti-RAP1GAP antibody [Y134] ab32373 staining at 1/10000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 90 kDa in Wild-type A549 cell lysates with no signal observed at this size in RAP1GAP knockout A549 cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

Lanes 1 - 4:

Western blot - Anti-RAP1GAP antibody [Y134] (ab32373) at 1/10000 dilution

Lanes 1 - 4:

Western blot - Anti-RAP1GAP antibody [Y134] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/rap1gap-antibody-y134-bsa-and-azide-free-ab247250'>ab247250</a>) at 1/10000 dilution

Lane 1:

Wild-type A549 at 20 µg

Lane 2:

RAP1GAP knockout A549 at 20 µg

Lane 3:

HT-29 at 20 µg

Lane 4:

Ramos at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 73 kDa

Observed band size: 90 kDa

false

• WB

Unknown

Western blot - Anti-RAP1GAP antibody [Y134] (AB32373)

The molecular weight observed is consistent with what has been described in the literature (PMID : 9346962, PMID : 15632203, PMID : 30144784).

All lanes:

Western blot - Anti-RAP1GAP antibody [Y134] (ab32373) at 1/50000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 73 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-RAP1GAP antibody [Y134] (AB32373)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 9346962, PMID : 15632203, PMID : 30144784).

All lanes:

Western blot - Anti-RAP1GAP antibody [Y134] (ab32373) at 1/10000 dilution

All lanes:

SH-SY5Y cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 73 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-RAP1GAP antibody [Y134] (AB32373)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 9346962, PMID : 15632203, PMID : 30144784).

All lanes:

Western blot - Anti-RAP1GAP antibody [Y134] (ab32373) at 1/10000 dilution

All lanes:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 73 kDa

Observed band size: 95 kDa

false

• WB

Lab

Western blot - Anti-RAP1GAP antibody [Y134] (AB32373)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 9346962, PMID : 15632203, PMID : 30144784).

All lanes:

Western blot - Anti-RAP1GAP antibody [Y134] (ab32373) at 1/10000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 73 kDa

Observed band size: 95 kDa

false