Anti-PAK4 antibody [EPR28630-82]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PAK4 KO HEK293T (human PAK4 knockout embryonic kidney epithelial cell, Left) / Parental HEK293T (Right) cells labelling PAK4 with ab314765 at 1/50 dilution (1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue labeling PAK4 with ab314765 at 1/50 (10.52 ug/ml) dilution, followed by a ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human colon carcinoma (PMID : 28542136). The section was incubated with ab314765 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Immunohistochemical analysis of paraffin-embedded (A) Parental HEK-293T (human embryonic kidney epithelial cell) cell pellet; (B) PAK4 knock-out HEK-293T cell pellet. Tissue labeling PAK4 with ab314765 at 1/500 (1.052 ug/ml) dilution, followed by a ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on (A) parental HEK-293T cell pellet, no staining on (B) PAK4 knock-out HEK-293T cell pellet. The section was incubated with ab314765 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue labeling PAK4 with ab314765 at 1/50 (10.52 ug/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human endometrial carcinoma. The section was incubated with ab314765 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling PAK4 with ab314765 at 1/50 (10.52 ug/ml) dilution, followed by a ready to use rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human breast carcinoma (PMID : 28407679). The section was incubated with ab314765 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IP

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Immunoprecipitation - Anti-PAK4 antibody [EPR28630-82] (AB314765)

PAK4 was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) whole cell lysate with ab314765 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314765 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : 293T (human embryonic kidney epithelial cell) whole cell lysate Lane 2 : ab314765 IP in 293T (human embryonic kidney epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314765 in 293T whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-PAK4 antibody [EPR28630-82] (ab314765) at 1/30 dilution

All lanes:

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

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 15s

• WB

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Western blot - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, ab314765 was shown to bind specifically to PAK4. Target of interest was observed at 68 kDa and 52 KD in wild-type HEK293T cell lysates (lane 1, wild-type cell line ab255449) with no signal observed at this size in PAK4 knockout cell line (lane 2, knockout cell line ab266807/ knockout cell lysate ab258560). The band (50 kDa) beneath the target band (68 kDa) is likely to be degraded target fragments. In western blot, anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-PAK4 antibody [EPR28630-82] (ab314765) at 1/1000 dilution

Lane 1:

Western blot - Human wild-type HEK-293T cell line (ab255449) at 20 µg

Lane 2:

Western blot - Human PAK4 knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-pak4-knockout-hek-293t-cell-lysate-ab258560'>ab258560</a>) at 20 µg

Lane 3:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Human testis lysate at 20 µg

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

Observed band size: 52 kDa,68 kDa

false

Exposure time: 70s

• WB

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Western blot - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. This target was tested in fresh and frozen LNCaP and Hela, and it was concluded that PAK4 is the easy target for degradation. Lane 3-5 was freshly made and used for western blotting immediately to minimize protein degradation. Exposure time : Lane 1, Lane 4-5 : 70 seconds; Lane 2 : 180 seconds; Lane 3 : 26 seconds

All lanes:

Western blot - Anti-PAK4 antibody [EPR28630-82] (ab314765) at 1/1000 dilution

Lanes 1 and 3:

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

Lanes 2 and 4:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

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

Observed band size: 52 kDa,68 kDa

false

• WB

Supplier Data

Western blot - Anti-PAK4 antibody [EPR28630-82] (AB314765)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. This antibody does not cross-react with human PAK5 and human PAK6. All the recombinant proteins were made in-house. In western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-PAK4 antibody [EPR28630-82] (ab314765) at 1/1000 dilution

Lane 1:

Purified His-tagged human PAK4 recombinant protein at 20 ng

Lane 2:

Purified His-tagged human PAK5 recombinant protein at 100 ng

Lane 3:

Purified His-tagged human PAK6 recombinant protein at 20 ng

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: 80 kDa

false

Exposure time: 48s