Anti-MST4 antibody [EP1864Y]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MST4 antibody [EP1864Y] (AB52491)

Overlay histogram showing HeLa cells stained with ab52491 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52491, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MST4 antibody [EP1864Y] (AB52491)

Immunohistochemical analysis of paraffin-embedded human placenta using ab52491 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MST4 antibody [EP1864Y] (AB52491)

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labelling MST4 with purified ab52491 at 1/500. Cells were fixed with 100% methanol and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• IP

Lab

Immunoprecipitation - Anti-MST4 antibody [EP1864Y] (AB52491)

MST4 was immunoprecipitated using ab52491 at 1 : 100 dilution (2μg in 0.35mg lysates).

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

All lanes:

Immunoprecipitation - Anti-MST4 antibody [EP1864Y] (ab52491) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

HeLa whole cell lysate immunoprecipitated with ab52491 1:100 dilution (2μg in 0.35mg lysates) at 10 µg

Lane 3:

HeLa whole cell lysate immunoprecipitated with Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) 1:100 dilution (2μg in 0.35mg lysates), at 10 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

Predicted band size: 47 kDa

false

Exposure time: 10s

• WB

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Western blot - Anti-MST4 antibody [EP1864Y] (AB52491)

All lanes:

Western blot - Anti-MST4 antibody [EP1864Y] (ab52491) at 1/200000 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 47 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-MST4 antibody [EP1864Y] (AB52491)

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

All lanes:

Western blot - Anti-MST4 antibody [EP1864Y] (ab52491) at 1/100000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue 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: 47 kDa

Observed band size: 50 kDa

false

Exposure time: 30s

• WB

Unknown

Western blot - Anti-MST4 antibody [EP1864Y] (AB52491)

ab52491 recognizes the full length tagged recombinant MST4 protein (ab60341) which has an expected molecular weight of 72 kDa.

All lanes:

Western blot - Anti-MST4 antibody [EP1864Y] (ab52491) at 1/20000 dilution

All lanes:

Western blot - Recombinant human MST4 protein (<a href='/en-us/products/proteins-peptides/recombinant-human-mst4-protein-ab60341'>ab60341</a>) at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 47 kDa

false

Exposure time: 20s