Anti-WTAP antibody [EPR18744]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-WTAP antibody [EPR18744] (AB195380)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling WTAP with ab195380 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on Jurkat cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab195380 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-WTAP antibody [EPR18744] (AB195380)

Immunohistochemical analysis of paraffin-embedded human liver cancer tissue labeling WTAP with ab195380 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human liver cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-WTAP antibody [EPR18744] (AB195380)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling WTAP with ab195380 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (Alexa Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-WTAP antibody [EPR18744] (AB195380)

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling WTAP with ab195380 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human endometrium is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-WTAP antibody [EPR18744] (AB195380)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling WTAP with ab195380 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab195380 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

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Immunoprecipitation - Anti-WTAP antibody [EPR18744] (AB195380)

WTAP was immunoprecipitated from 0.35 mg of Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate with ab195380 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab195380 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Jurkat whole cell lysate 10μg (Input).

Lane 2 : ab195380 IP in Jurkat whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab195380 in Jurkat whole cell lysate.

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

Exposure time : 30 seconds.

All lanes:

Immunoprecipitation - Anti-WTAP antibody [EPR18744] (ab195380)

Predicted band size: 44 kDa

false

• WB

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Western blot - Anti-WTAP antibody [EPR18744] (AB195380)

In Western blot, Anti-WTAP antibody [EPR18744] - Loading Control (ab195380) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-WTAP (phospho S306) antibody [EPR27038-188] (<a href='/en-us/products/primary-antibodies/wtap-phospho-s306-antibody-epr27038-188-ab316275'>ab316275</a>) at 1/1000 dilution

Lane 1:

Jurkat (human t cell leukemia t lymphocyte from peripheral blood) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Jurkat 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 1/20000 dilution

Observed band size: 55 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-WTAP antibody [EPR18744] (AB195380)

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] (<a href='/en-us/products/primary-antibodies/wtap-phospho-s341-antibody-epr27039-57-ab309350'>ab309350</a>) 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

• WB

Supplier Data

Western blot - Anti-WTAP antibody [EPR18744] (AB195380)

In Western blot, Anti-WTAP antibody [EPR18744] - Loading Control (ab195380) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-WTAP (phospho S306) antibody [EPR27038-188] (<a href='/en-us/products/primary-antibodies/wtap-phospho-s306-antibody-epr27038-188-ab316275'>ab316275</a>) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

A375 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 1/20000 dilution

Observed band size: 55 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-WTAP antibody [EPR18744] (AB195380)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : WTAP knockout HAP1 whole cell lysate (20 μg)
Lane 3 : MOLT-4 whole cell lysate (20 μg)
Lane 4 : K562 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab195380 observed at 50 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab195380 was shown to specifically react with WTAP in wild-type HAP1 cells as signal was lost in WTAP knockout cells. Wild-type and WTAP knockout samples were subjected to SDS-PAGE. ab195380 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-WTAP antibody [EPR18744] (ab195380)

Predicted band size: 44 kDa

false

• WB

Supplier Data

Western blot - Anti-WTAP antibody [EPR18744] (AB195380)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed molecular weight is consistent with what has been described in the literature (PMID : 17088532).

All lanes:

Western blot - Anti-WTAP antibody [EPR18744] (ab195380) at 1/1000 dilution

Lane 1:

Human kidney lysate at 10 µg

Lane 2:

Human thymus lysate at 10 µg

Lane 3:

Human lung lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 44 kDa

Observed band size: 50 kDa

true

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-WTAP antibody [EPR18744] (AB195380)

Blocking/Dilution buffer : 5% NFDM/TBST.

The observed molecular weight is consistent with what has been described in the literature (PMID : 17088532).

All lanes:

Western blot - Anti-WTAP antibody [EPR18744] (ab195380) at 1/1000 dilution

Lane 1:

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

Lane 2:

K562 (Human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate at 20 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

HepG2 (Human liver hepatocellular carcinoma cell line) 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

Predicted band size: 44 kDa

Observed band size: 50 kDa

true

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-WTAP antibody [EPR18744] (AB195380)

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] (<a href='/en-us/products/primary-antibodies/wtap-phospho-s341-antibody-epr27039-57-ab309350'>ab309350</a>) 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