Anti-PAK2 抗体 [EP796Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (AB76293)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PAK2 with purified ab76293 at 1 : 100 dilution (2.0μg/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) 1 : 200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PAK2 antibody [EP796Y] (AB76293)

Overlay histogram showing HeLa cells stained with unpurified ab76293 (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 (ab76293, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PAK2 antibody [EP796Y] (AB76293)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PAK2 with purified ab76293 at 1/20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK2 antibody [EP796Y] (AB76293)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling PAK2 with Purified ab76293 at 1 : 100 dilution (2.02 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PAK2 antibody [EP796Y] (AB76293)

ICC/IF image of unpurified ab76293 stained T47D cells. The cells were 100% methanol fixed (5 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 (ab76293, 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.

• IP

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Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (AB76293)

PAK2 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate 10 µg with ab76293 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab76293 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) cell lysate 10 µg

Lane 2 : ab76293 IP in HeLa cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab76293 in HeLa cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 7 seconds

All lanes:

Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)

Predicted band size: 58 kDa

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• IP

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Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (AB76293)

PAK2 was immunoprecipitated from 0.35 mg NIH/3T3 (Mouse embryonic fibroblast) cell lysate 10 µg with ab76293 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab76293 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) cell lysate 10 µg

Lane 2 : ab76293 IP in NIH/3T3 cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab76293 in HeLa cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 7 seconds

All lanes:

Immunoprecipitation - Anti-PAK2 antibody [EP796Y] (ab76293)

Predicted band size: 58 kDa

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• WB

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Western blot - Anti-PAK2 antibody [EP796Y] (AB76293)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : PAK2 knockout HAP1 cell lysate (20 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : Human lymph node tissue lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab76293 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab76293 was shown to specifically react with PAK2 when PAK2 knockout samples were used. Wild-type and PAK2 knockout samples were subjected to SDS-PAGE. ab76293 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-PAK2 antibody [EP796Y] (ab76293)

Predicted band size: 58 kDa

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• WB

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Western blot - Anti-PAK2 antibody [EP796Y] (AB76293)

All lanes:

Western blot - Anti-PAK2 antibody [EP796Y] (ab76293) at 1/2000 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 58 kDa

Observed band size: 61 kDa

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• WB

Lab

Western blot - Anti-PAK2 antibody [EP796Y] (AB76293)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PAK2 antibody [EP796Y] (ab76293) at 1/5000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 3:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Lane 4:

C6 (Rat glial tumor glial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 58 kDa

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• WB

Lab

Western blot - Anti-PAK2 antibody [EP796Y] (AB76293)

Lanes 1- 2 : Merged signal (red and green). Green - ab76293 observed at 60 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab76293 was shown to react with PAK2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264814 (knockout cell lysate ab257573) was used. Wild-type HeLa and PAK2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab76293 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PAK2 antibody [EP796Y] (ab76293) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PAK2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PAK2 knockout HeLa cell line (<a href='/products/cell-lines/human-pak2-knockout-hela-cell-line-ab264814'>ab264814</a>)

Predicted band size: 50 kDa,58 kDa

Observed band size: 50 kDa,60 kDa

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• WB

Supplier Data

Western blot - Anti-PAK2 antibody [EP796Y] (AB76293)

False colour image of Western blot : Anti-PAK2 antibody [EP796Y] staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab76293 was shown to bind specifically to PAK2. A band was observed at 65 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in PAK2 CRISPR-Cas9 edited cell line ab282648 (CRISPR-Cas9 edited cell lysate ab283047). The band observed in the CRISPR-Cas9 edited lysate lane below 65 kDa is likely to represent a truncated form of PAK2. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and PAK2 CRISPR-Cas9 edited HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PAK2 antibody [EP796Y] (ab76293) at 1/5000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human PAK2 knockout HEK-293T cell lysate (ab283047) at 20 µg

Lane 3:

Wild-type HeLa ab255552 cell lysate at 20 µg

Lane 4:

PAK2 knockout HeLa ab260287 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 58 kDa

Observed band size: 65 kDa

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