Anti-OSMR antibody [RM2085]

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-OSMR antibody [RM2085] (AB324099)

Flow cytometric analysis of A-172 (human brain glioblastoma cell) (Right) / JAR (human placenta choriocarcinoma epithelial cell) (Left) cells labelling OSMR with ab324099 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Negative control : JAR.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-OSMR antibody [RM2085] (AB324099)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized A375 (human malignant melanoma epithelial cell) cells labelling OSMR with ab324099 at 1/50 (9.94 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly membranous staining in A375 and no staining in Jurkat cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : Jurkat (PMID : 8999038).
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 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 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-OSMR antibody [RM2085] (AB324099)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized A-172 (human brain glioblastoma cell) cells labelling OSMR with ab324099 at 1/50 (9.94 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing membranous and cytoplasmic staining in A-172 and no staining in JAR cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : JAR (PMID : 8999038).
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 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 dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-OSMR antibody [RM2085] (AB324099)

Flow cytometric analysis of A375 (human malignant melanoma epithelial cell) (Right) / Jurkat (human T cell leukemia T lymphocyte from peripheral blood) (Left) cells labelling OSMR with ab324099 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Negative control : Jurkat.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-OSMR antibody [RM2085] (AB324099)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (Human embryonic kidney epithelial cell) cells labelling OSMR with ab324099 at 1/50 (9.94 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing positive staining in 293T cells transfected with a human OSMR expression vector containing a myc-His-tag® (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).

Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 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 dilution.

• IP

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Immunoprecipitation - Anti-OSMR antibody [RM2085] (AB324099)

OSMR was immunoprecipitated from 0.35 mg ab324099 at 1/30 IP in A375 (human malignant melanoma epithelial cell) whole cell lysate with ab324099 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324099 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-OSMR antibody [RM2085] (ab324099) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab324099 at 1/30 IP in A375 (human malignant melanoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324099 in A375 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 150-180 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-OSMR antibody [RM2085] (AB324099)

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

Negative control : JAR, Jurkat (PMID : 8999038, 8119965, 28607076).

Low expression : human tonsil (PMID : 8999038).

The expression pattern/observed MW are consistent with what has been described in the literature (PMID : 8999038, 9188471, 21394648).

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

All lanes:

Western blot - Anti-OSMR antibody [RM2085] (ab324099) at 1/1000 dilution

Lane 1:

HaCaT (human skin keratinocyte) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

A-172 (human brain glioblastoma cell ) whole cell lysate at 20 µg

Lane 5:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 6:

JAR (human placenta choriocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

Human lung cancer tissue lysate at 20 µg

Lane 8:

Human tonsil tissue lysate at 20 µg

Secondary

Lanes 1 - 6:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Lanes 7 - 8:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 150-180 kDa,36 kDa

false

Exposure time: 180s