Anti-PAK4 antibody [EPR25195-94]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAK4 antibody [EPR25195-94] (AB300505)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MCF7 (human breast adenocarcinoma epithelial) cells labelling PAK4 with ab300505 at 1/50 (9.52 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/mL dilution (Green). Confocal image showing mostly cytoplasmic staining in MCF-7 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/mL dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAK4 antibody [EPR25195-94] (AB300505)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PAK4 KO HEK293T (ab258560) cells labelling PAK4 with ab300505 at 1/50 (9.52 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/mL dilution (Green). Confocal image showing mostly cytoplasmic staining in parental HEK293T cell line, and no staining in PAK4 KO HEK293T cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/mL dilution.

• WB

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Western blot - 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.

All lanes:

Western blot - Anti-PAK4 antibody [EPR25195-94] (ab300505) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

PAK4 knockout HEK-293T whole cell lysate at 20 µg

Lane 3:

LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 64 kDa

Observed band size: 68 kDa

false

• WB

Supplier Data

Western blot - Anti-PAK4 antibody [EPR25195-94] (AB300505)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-PAK4 antibody [EPR25195-94] (ab300505) at 1/1000 dilution

Lane 1:

LNCaP (human prostate carcinoma epithelial cell), whole cell lysate

Lane 2:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell), whole cell lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 68 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-PAK4 antibody [EPR25195-94] (AB300505)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-PAK4 antibody [EPR25195-94] (ab300505) at 1/1000 dilution

Lane 1:

Purified His-tagged human PAK4 recombinant protein

Lane 2:

Purified His-tagged human PAK5 recombinant protein

Lane 3:

Purified His-tagged human PAK6 recombinant protein

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 70 kDa

false

Exposure time: 26s