Anti-CD90 / Thy1 antibody [EPR28145-53]

• Flow Cyt

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Flow Cytometry - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Flow cytometric analysis of K-562 (human chronic myelogenous leukemia lymphoblast) (Left panel) compared to U-2 OS (human bone osteosarcoma epithelial cell) (Right panel) labeling CD90 / Thy1 with ab307736 at 1/500 dilution (0.1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Negative control : K-562.

Gated on viable cells.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS (human bone osteosarcoma epithelial cell) cells labeling CD90 / Thy1 with ab307736 at 1/250 dilution (1.8 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing membranous and cytoplasmic staining in U-2 OS cell line, no staining was observed in K-562 cell line. Negative control : K-562 (PMID : 7683034). 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 (2.5 µg/ml) (Red). 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 (2 µg/ml).

• IP

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Immunoprecipitation - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

CD90 / Thy1 was immunoprecipitated from 0.35 mg U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate 10 µg with ab307736 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307736 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : U-2 OS whole cell lysate 10 µg Lane 2 : ab307736 IP in U-2 OS whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307736 in HU-2 OS whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 15 seconds. The molecular weight observed is consistent with what has been described in the literature (PMID : 16770003) and due to the high glycosylation of the protein.

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• IP

Supplier Data

Immunoprecipitation - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

CD90 / Thy1 was immunoprecipitated from 0.35 mg HuT-78 (human sezary syndrome cutaneous t lymphocyte) whole cell lysate 10 µg with ab307736 at 1/30 dilution (2 µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307736 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HuT-78 whole cell lysate 10 µg Lane 2 : ab307736 IP in HuT-78 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307736 in HuT-78 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 15 seconds. The molecular weight observed is consistent with what has been described in the literature (PMID : 16770003) and due to the high glycosylation of the protein.

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized EL4 (mouse lymphoma T lymphocyte) cells labeling CD90 / Thy1 with ab307736 at 1/250 dilution (1.8 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing membranous and cytoplasmic staining in EL4 cell line. 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 (2.5 µg/ml) (Red). 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 (2 µg/ml).

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Flow cytometric analysis of Rabbit monoclonal IgG isotype control (ab172730) (Left panel) compared to mouse peripheral blood mononuclear cell (PBMC) (Right panel) labeling CD90 / Thy1 with ab307736 at 1/500 dilution (0.1 µg). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Cells were stained with rabbit IgG or ab307736 then stained with anti-CD3 conjugated to Alexa Fluor® 647.

Gated on viable cells.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Flow cytometric analysis of PC-12 (rat adrenal gland pheochromocytoma) cells labeling CD90 / Thy1 with ab307736 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG isotype control (ab172730) (Black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma small irregularly shaped cells) cells labeling CD90 / Thy1 with ab307736 at 1/250 dilution (1.8 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing membranous and cytoplasmic staining in PC-12 cell line. 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 (2.5 µg/ml) (Red). 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 (2 µg/ml).

• WB

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Western blot - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The molecular weight observed is consistent with what has been described in the literature  (PMID : 16770003) and due to the highglycosylation of the protein. Exposure time : 48 seconds.

All lanes:

Western blot - Anti-CD90 / Thy1 antibody [EPR28145-53] (ab307736) at 1/1000 dilution

Lane 1:

Human spleen tissue lysate at 20 µg

Lane 2:

HuT-78 (human sezary syndrome cutaneous t lymphocyte) whole cell lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

PC-12 (rat adrenal gland pheochromocytoma 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

Observed band size: 25 kDa,37 kDa

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Exposure time: 48s

• WB

Lab

Western blot - Anti-CD90 / Thy1 antibody [EPR28145-53] (AB307736)

Western blot : Anti-THY1 antibody [EPR28145-53] (ab307736) staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab307736 was shown to bind specifically to THY1. A band was observed at 25-37 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in THY1 knockout cell line ab262490 (knockout cell lysate ab263925). To generate this image, wild-type and THY1 knockout U-2 OS cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CD90 / Thy1 antibody [EPR28145-53] (ab307736) at 1/5000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 30 µg

Lane 2:

Western blot - Human THY1 (CD90) knockout U-2 OS cell lysate (<a href='/en-us/products/cell-lysates/human-thy1-cd90-knockout-u-2-os-cell-lysate-ab263925'>ab263925</a>)

Lane 2:

Western blot - Human THY1 (CD90) knockout U-2 OS cell line (<a href='/en-us/products/cell-lines/human-thy1-cd90-knockout-u-2-os-cell-line-ab262490'>ab262490</a>)

Lane 2:

THY1 knockout U-2 OS cell lysate at 30 µg

Lane 3:

Human brain cell lysate at 2 µg

Lane 4:

Human kidney cell lysate at 20 µg

Lane 5:

K562 cell lysate at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 17 kDa

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