重组Anti-MST抗体[EPR29168-60] (ab317833)

Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

The samples were run on a Bis-Tris gel under reducing conditions.

Western blot: Anti-MST antibody (ab317833) staining at 1/1000 dilution, shown in green; Mouse anti-alpha Tubulin antibody [DM1A] (ab7291) loading control staining at 1/20, 000 dilution, shown in magenta.

In Western blot, ab317833 was shown to bind specifically to MST. Target of interest was observed at 33 kDa and 35 KDa in wild-type HEK-293T cell lysates (lane 1) with no signal observed at these sizes in MPST (MST) knockout cell line (lane 2, knockout cell line ab266789 / knockout cell lysate ab258524). 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 fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C.  Blots were washed 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.

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

Low expression: MOLT-4 (PMID: 35204649).

The identity of the lower MW band at approximately 27 kDa (in lane 1) is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

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

Low expression: lung (PMID: 29712741).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Exposure time: Lanes 1-5: 1 second; Lanes 6-8: 3 seconds.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling MST with ab317833 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human liver. The section was incubated with ab317833 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 10 mins

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling MST with ab317833 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The section was incubated with ab317833 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 10 mins

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling MST with ab317833 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse liver (PMID:©27973427). The section was incubated with ab317833 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 10 mins

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling MST with ab317833 at 1/2000 (0.255 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat liver. The section was incubated with ab317833 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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 Citrate buffer (pH 6.0, Epitope Retrieval Solution 1) for 10 mins

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized MPST (MST) KO HEK-293T (MPST(MST) knockout human embryonic kidney epithelial cell), ab266789 cells labelling MST with ab317833 at 1/500 (1.018 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing mainly cytoplasmic staining in wildtype HEK-293T cells and negative staining in MPST (MST) knockout HEK-293T cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized A431 (human epidermoid carcinoma epithelial cell) cells labelling MST with ab317833 at 1/500 (1.018 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing mainly cytoplasmic staining in A431 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HEK293T (human embryonic kidney epithelial cell, Left) / MPST(MST) knockout HEK293T (Right) cells labelling MST with ab317833 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A431 (human epidermoid carcinoma epithelial cell) cells labelling MST with ab317833 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

MST was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) whole cell lysate with ab317833 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317833 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2: ab317833 IP in 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317833 in 293T whole cell lysate

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

Exposure time: 24 seconds..

Dot blot analysis of MST using ab317833 at 1:1000 (0.509 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

Lane 1: His-tagged human MST fragment.

Lane 2: His-tagged human TST fragment.

Exposure time: 3 seconds.

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

This antibody does not cross-react with human TST.

Anti-His tag® antibody (1:10000) as total protein control.