Anti-WSTF antibody [EP1704Y]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-WSTF antibody [EP1704Y] (AB51256)

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling WSTF with Purified ab51256 at 1 : 20 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1 : 2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-WSTF antibody [EP1704Y] (AB51256)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling WSTF with Purified ab51256 at 1 : 700 dilution (0.211 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-WSTF antibody [EP1704Y] (AB51256)

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling WSTF with Purified ab51256 at 1 : 50 dilution (2.9 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-WSTF antibody [EP1704Y] (AB51256)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cardiac muscle tissue sections labeling WSTF with Purified ab51256 at 1 : 700 dilution (0.211 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-WSTF antibody [EP1704Y] (AB51256)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cardiac muscle tissue sections labeling WSTF with Purified ab51256 at 1 : 700 dilution (0.211 µg/ml). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• WB

Unknown

Western blot - Anti-WSTF antibody [EP1704Y] (AB51256)

Exposure time :

Left image : 180 seconds

Right image : 40 seconds

We recommend to use ab109439 for WSTF Western Blot testing because ab51256 detects nonspecific bands.

All lanes:

Western blot - Anti-WSTF antibody [EP1704Y] (ab51256) at 1/20000 dilution

Lane 1:

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

Lane 2:

T-47D (Human ductal breast epithelial tumor epithelial cell) whole cell lysate at 20 µg

Lane 3:

MCF7 (Human breast adenocarcinoma 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

Predicted band size: 171 kDa

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• WB

Lab

Western blot - Anti-WSTF antibody [EP1704Y] (AB51256)

Lanes 1- 2 : Merged signal (red and green). Green - ab51256 observed at 175 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab51256 was shown to react with WSTF in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264907 (knockout cell lysate ab257370) was used. Wild-type HeLa and WSTF knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab51256 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 15000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-WSTF antibody [EP1704Y] (ab51256) at 1/15000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

BAZ1B knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human BAZ1B (WSTF) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-baz1b-wstf-knockout-hela-cell-line-ab264907'>ab264907</a>)

Predicted band size: 171 kDa

Observed band size: 175 kDa

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• WB

Unknown

Western blot - Anti-WSTF antibody [EP1704Y] (AB51256)

Lanes 1 - 4 : Merged signal (red and green). Green - ab51256 observed at 175 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab51265 was shown to recognize WSTF in wilt-type cells along with additional cross-reactive bands as signal was lost in WSTF knockout samples. Wild-type and WSTF knockout samples were subjected to SDS-PAGE. ab51256 and ab18058 (loading control to vinculin) were diluted 1/15 000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-WSTF antibody [EP1704Y] (ab51256) at 1/15000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

WSTF knockout HAP1 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

PC12 cell lysate at 20 µg

Predicted band size: 171 kDa

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• WB

Unknown

Western blot - Anti-WSTF antibody [EP1704Y] (AB51256)

Exposure time

Lane 1 : 10 seconds

Lane 2 : 40 seconds

Lane 3 : 180 seconds

All lanes:

Western blot - Anti-WSTF antibody [EP1704Y] (ab51256) at 1/5000 dilution

Lane 1:

B16-F0 (Mouse melanoma epithelial cell-like) whole cell lysate at 15 µg

Lane 2:

Mouse testis lysate at 15 µg

Lane 3:

Rat testis lysate at 15 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

Predicted band size: 171 kDa

Observed band size: 185 kDa

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• WB

CiteAb

Western blot - Anti-WSTF antibody [EP1704Y] (AB51256)

WSTF western blot using anti-WSTF antibody [EP1704Y] ab51256. Publication image and figure legend from Liu, Y., Wang, S. Q., et al., 2016, Oncotarget, PubMed 27449290.

ab51256 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab51256 please see the product overview.

Release of WSTF was induced by KRASG12V in colon cells(A) We constructed the H-RasG12V expression vectors to preferentially activate the Ras pathways. The levels of P-ERK1/2, P-AKT and RalA-GTP were detected with specific antibodies to examine the activities of corresponding pathways. (B) Media containing WSTF and P-WSTF was detected using ELISA. Levels of intracellular P-WSTF, total WSTF protein and mRNA were detected as controls. (C) Different small interfering RNAs were transfected into HIPECKRASM cells for 48 h. Two specific siRNAs for each gene were implicated in experiments. The secreted WSTF was detected by ELISA assay. (D) The media of HIPECKRASM cells, which was cultured with U0126 (10 μM) or Wortmannin (10 μM) for 20 h, were tested by ELISA. Non-treated HIPECKRASM cells were used as control.

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