Anti-PAK2 antibody [EP796Y]

12 product images

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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)(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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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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  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 (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 Human PAK2 knockout HeLa cell line ab264814 (knockout cell lysate Human PAK2 knockout HeLa 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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 (Human PAK2 knockout HeLa cell line ab264814)

  Performed under reducing conditions.

  Predicted band size: 50 kDa, 58 kDa

  Observed band size: 50 kDa, 60 kDa

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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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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.

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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)(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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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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  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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.

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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).
  

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  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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  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, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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

• 

  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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  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 Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed 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.

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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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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.
  

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  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] (Anti-GAPDH antibody [6C5] - Loading Control 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