Recombinant Anti-PAK1+PAK2+PAK3 (phospho S144 + S154 + S144 + S141) antibody [EP656Y] (ab40795)

Blocking and dilution buffer: 5% NFDM/TBST.

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in rat cerebral cortex tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in HeLa (human cervix adenocarcinoma) cells, treated and untreated with Lambda Protein Phosphtase 31℃ for 5h by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody. ab7291 and ab150120 were used as counterstains for primary antibody ab75748 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in the human cell line NIH/3T3 (mouse embryo) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/120. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/500 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

ab40795 immunoprecipitating PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154). 10µg of HeLa (human cervix adenocarcinoma) whole cell lysate was incubated with primary antibody at a dilution of 1/40 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1: HeLa whole cell lysate (10ug)
Lane 2: ab40795 IP in HeLa whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab40795 in HeLa (human cervix adenocarcinoma) whole cell lysate

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in mouse cerebral cortex tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

Overlay histogram showing HeLa cells stained with unpurified ab40795 (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 (ab40795, 1/100 dilution) 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. 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.

ab40795 staining PAK1 (phospho S144) + PAK2 (phospho S141) + PAK3 (phospho S154) in human liver carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.