Recombinant Anti-PAK4 antibody [EPR25195-94] (ab300505)

False colour image of Western blot: Anti-PAK4 antibody (ab300505) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab300505 was shown to bind specifically to PAK4. A band was observed at 68 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in PAK4 knockout cell line ab266807 (knockout cell lysate ab258560). To generate this image, wild-type and PAK4 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

The bands beneath the target band (68 kDa) are expected to be degradation products

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

Exposure time: 10 seconds. 

Lysates were freshly made and used immediately for Western blotting to minimize protein degradation.

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

Exposure time: 26 seconds. 

This antibody does not cross-react with human PAK5 and human PAK6.
All the recombinant proteins were made in-house and expressed from the E. coli expression systems.

Immunofluorescent analysisis of 4% papraformaldehyde fixed and 0.1% TritonX-100 permeabilized MCF7 (human breast adenocarcinoma epithelial cell）labeling PAK4 with ab300505 at 1/50 dilution, followed by preadsorbed Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, Green, ab150081) at 1/1000 dilution. Confocal image showing mostly cytoplasmic staining in MCF-7 cell line. 

Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594, Red, ab195889) was used as counterstain at 1/200 dilution. Nuclear counterstain is DAPI. 

Secondary antibody control: PBS was used instead of primary antibody, followed by ready to preadsorbed Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, Green, ab150081) at 1/1000 dilution. 

Immunofluorescent analysisis of 4% papraformaldehyde fixed and 0.1% TritonX-100 permeabilized PAK4 KO HEK293T (ab258560) or parental HEK293T cell line labeling PAK4 with ab300505 at 1/50 dilution, followed by preadsorbed Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, Green, ab150081) at 1/1000 dilution. Confocal image showing mostly cytoplasmic staining in parental HEK293T cell line, and no staining in PAK4 KO HEK293T cell line. 

Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594, Red, ab195889) was used as counterstain at 1/200 dilution. Nuclear counterstain is DAPI. 

Secondary antibody control: PBS was used instead of primary antibody, followed by ready to preadsorbed Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, Green, ab150081) at 1/1000 dilution.