Anti-PAK4 antibody [EPR25195-73] (BSA and Azide free)

• WB

Supplier Data

Western blot - Anti-PAK4 antibody [EPR25195-73] (BSA and Azide free) (AB300504)

This data was developed using ab300503, the same antibody clone in a different buffer formulation.

False colour image of Western blot : Anti-PAK4 antibody [EPR25195-73] (ab300503) 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, ab300503 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 [EPR25195-73] (<a href='/en-us/products/primary-antibodies/pak4-antibody-epr25195-73-ab300503'>ab300503</a>) 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 [EPR25195-73] (BSA and Azide free) (AB300504)

This data was developed using ab300503, the same antibody clone in a different buffer formulation. Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-PAK4 antibody [EPR25195-73] (<a href='/en-us/products/primary-antibodies/pak4-antibody-epr25195-73-ab300503'>ab300503</a>) 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: 68 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-PAK4 antibody [EPR25195-73] (BSA and Azide free) (AB300504)

This data was developed using ab300503, the same antibody clone in a different buffer formulation. Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-PAK4 antibody [EPR25195-73] (<a href='/en-us/products/primary-antibodies/pak4-antibody-epr25195-73-ab300503'>ab300503</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate

Lane 4:

THP-1 (human monocytic leukemia monocyte) whole cell lysate

Lane 5:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate

Lane 6:

NR8383 (rattus norvegicus lu macrophage (alveolar)) whole cell lysate 20

Lane 7:

C2C12 (mouse myoblasts myoblast), whole cell lysate

Lane 8:

LNCaP (human prostate 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

• WB

Supplier Data

Western blot - Anti-PAK4 antibody [EPR25195-73] (BSA and Azide free) (AB300504)

This data was developed using ab300503, the same antibody clone in a different buffer formulation.

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

We recommend using freshly made lysate to perform western blot testing.

All lanes:

Western blot - Anti-PAK4 antibody [EPR25195-73] (<a href='/en-us/products/primary-antibodies/pak4-antibody-epr25195-73-ab300503'>ab300503</a>) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) whole cell lysate, frozen lysate at 20 µg

Lane 2:

293T (Human embryonic kidney epithelial cell) whole cell lysate, fresh lysate

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate, frozen lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate, fresh 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 1/20000 dilution

Predicted band size: 64 kDa

Observed band size: 68 kDa

false

Exposure time: 40s