Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (AB51134)

Intracellular Flow Cytometry analysis of HeLa cells labelling Serine/theronine-protein kinase 4/MST-1 with purified ab51134 at a dilution of 1/50 (red). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. An Alexa Flour^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (AB51134)

ab51134 staining Serine/threonine-protein kinase 4/MST-1 in human gastric carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/50. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1 : PBS in place of primary antibody.

• IP

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Immunoprecipitation - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (AB51134)

ab51134 immunoprecipitating Serine/threonine-protein kinase 4/MST-1. 10μg of cell lysate was incubated with primary antibody at a dilution of 1/30 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/1000.

Lane 1 : Jurkat (human acute T cell leukemia) whole cell lysate (10ug)
Lane 2 : Jurkat (human acute T cell leukemia) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab51134 in Jurkat (human acute T cell leukemia) whole cell lysate

All lanes:

Immunoprecipitation - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (ab51134)

Predicted band size: 55 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (AB51134)

ab51134 staining Serine/threonine-protein kinase 4/MST-1 in Raw264.7 (mouse abelson murine leukemia virus-induced tumor) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a concentration of 1/1000. ab7291 anti-Tubulin (mouse mAb) (1/1000) and ab150120 AlexaFluor®594 Goat anti-Mouse secondary (1/1000) were used as counterstains for primary antibody ab51134 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1 : Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2 : Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

• WB

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Western blot - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (AB51134)

Lanes 1-4 : Merged signal (red and green). Green - ab51134 observed at 52 kDa. Red - loading control ab8245 observed at 37 kDa.

ab51134 Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] was shown to specifically react with MST-1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265442 (knockout cell lysate ab258215) was used. Wild-type and MST-1 knockout samples were subjected to SDS-PAGE. ab51134 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (ab51134) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

STK4 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human STK4 (Serine/threonine-protein kinase 4/MST-1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-stk4-serine-threonine-protein-kinase-4-mst-1-knockout-hela-cell-line-ab265442'>ab265442</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

Ramos cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 55 kDa

Observed band size: 52 kDa

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• WB

Lab

Western blot - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (AB51134)

Diluting and blocking buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (ab51134) at 1/50000 dilution

Lane 1:

Jurkat (human acute T cell leukemia) whole cell lysate at 1/20 dilution

Lane 2:

Mouse spleen at 1/20 dilution

Lane 3:

Rat spleen at 1/20 dilution

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 55 kDa

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• WB

Lab

Western blot - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (AB51134)

Diluting and blocking buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Serine/threonine-protein kinase 4/MST-1 antibody [EP1465Y] (ab51134) at 1/10000 dilution

All lanes:

HeLa (human cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 55 kDa

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