Recombinant 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.

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

Negative control: K-562 (PMID: 7683034).

The molecular weight observed is consistent with what has been described in the literature (PMID: 16770003).

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200, 000 dilution.

This blot was developed using a high sensitivity ECL substrate.

Exposure time: 103 seconds.

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.

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.

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 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.

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.

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.

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).

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).

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).