All lanes : Anti-PAK2 antibody [EP796Y] (ab76293) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PAK2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (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.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

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 Fluor^® 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). 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

This WB data was generated using the same anti-PAK2 antibody clone, EP796Y, in a different buffer formulation (cat# 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.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

This IHC data was generated using the same anti-PAK2 antibody clone, EP796Y, in a different buffer formulation (cat# ab76293).

Unpurified ab76293, at a 1/100 dilution, staining PAK2 in paraffin embedded human breast carcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).