Anti-WTAP (phospho S341) antibody [EPR27039-57]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-WTAP (phospho S341) antibody [EPR27039-57] (AB309350)

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling WTAP (phospho S341) with ab309350 at 1/500 (1.044 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human endometrium without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab309350 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrumentIncubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). 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-WTAP (phospho S341) antibody [EPR27039-57] (AB309350)

Immunohistochemical analysis of paraffin-embedded Human endometrial ca tissue labeling WTAP (phospho S341) with ab309350 at 1/500 (1.044 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human endometrial carcinoma without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B).The section was incubated with ab309350 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). 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-WTAP (phospho S341) antibody [EPR27039-57] (AB309350)

WTAP (phospho S341) was immunoprecipitated from 0.35 mg Parental HAP1 (wildtype control human chronic myelogenous leukemia near-haploid cell line) whole cell lysate with ab309350 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab309350 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Parental HAP1 (wildtype control human chronic myelogenous leukemia near-haploid cell line) whole cell lysate 4 ug
Lane 2 : ab309350 IP in Parental HAP1 (wildtype control human chronic myelogenous leukemia near-haploid cell line) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab309350 in parental HAP1whole cell lysate

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

Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-WTAP (phospho S341) antibody [EPR27039-57] (ab309350) at 1/1000 dilution

All lanes:

Parental HAP1 whole cell lysate at 4 µg

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

Observed band size: 55 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-WTAP (phospho S341) antibody [EPR27039-57] (AB309350)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-WTAP (phospho S341)antibody (ab309350) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 (ab8245) loading control staining at 1/20000 dilution, shown in red. The identity of the lower MW at approximately 35KD is unknown. To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescentwestern blot (TBS-based) blocking solution before incubation with primaryantibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-WTAP (phospho S341) antibody [EPR27039-57] (ab309350) at 1/1000 dilution

All lanes:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-ab97053'>ab97053</a>) at 1/100000 dilution

Observed band size: 55 kDa

false

• WB

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Western blot - Anti-WTAP (phospho S341) antibody [EPR27039-57] (AB309350)

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti-WTAP antibody (ab195380) as pan control staining at 1/1000 dilution.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-WTAP (phospho S341) antibody [EPR27039-57] (ab309350) at 1/1000 dilution

Lane 1:

Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Wild-type HAP1 whole cell lysate (Alkaline phosphatase treated membrane) 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 1/100000 dilution

Observed band size: 55 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-WTAP (phospho S341) antibody [EPR27039-57] (AB309350)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression of WTAP (phospho S341) is upregulated in response to PMA treatment (PMID : 33217317). In Western blot, anti-WTAP antibody (ab195380) as pan control staining at 1/1000 dilution. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-WTAP (phospho S341) antibody [EPR27039-57] (ab309350) at 1/1000 dilution

Lane 1:

Untreated Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 20 µg

Lane 2:

HAP1 treated with /ml PMA for 30 minutes whole cell lysate at 20 µg

Lane 3:

Untreated WTAP knockout HAP1 whole cell lysate at 20 µg

Lane 4:

WTAP knockout HAP1 treated with /ml PMA for 30 minutes 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 1/100000 dilution

Observed band size: 55 kDa

false

Exposure time: 180s

• Dot

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Dot Blot - Anti-WTAP (phospho S341) antibody [EPR27039-57] (AB309350)

Dot blot analysis of WTAP (phospho S341) using ab309350 at 1 : 1000 (0.522 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Exposure time : 180 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST