Anti-CD47 抗体 [EPR24922-21] (ab300124)

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

Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Samples are non-boiled as boiling may cause protein aggregates.
False colour image of Western blot: Anti-CD47 antibody [EPR24922-21] (ab300124) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab300124 was shown to bind specifically to CD47. A diffuse band was observed at 47-52 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in CD47 knockout cell line ab266324 (knockout cell lysate ab257220). To generate this image, wild-type and CD47 knockout HEK-293T cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
Negative control: HepG2 (PMID: 28378740)

Blocking / Diluting buffer and concentration:

5% NFDM/TBST

Negative control: HepG2 (PMID: 28378740)
Samples are non-boiled as boiling may cause protein aggregates.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeilized Jurkat (Human T cell leukemia T lymphocyte) cells lebelling CD47 with ab300124 at 1/100 (4.82 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing membranous staining in Jurkat cell line. Negative control: HepG2 (PMID: 28378740). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (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 (2 µg/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeilized CD47 KO HEK293T ( CD47 knockout human embryonic kidney epithelial cell) (ab266324) cells lebelling CD47 with ab300124 at 1/100 (4.82 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing membranous staining in Parental HEK293T cell line, no staining in CD47 KO HEK293T cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (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 (2 µg/ml) dilution.

Flow cytometric analysis of Wild-type HEK-293T (human embryonic kidney epithelial cell, Right) / CD47 knockout HEK-293T (Left) cells labelling CD47 with ab300124 at 1/500 dilution (0.1µg) (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 1/2000 dilution was used as the secondary antibody. Gated on viable cells. Positive staining on HEK-293T cells (ab255449), while no staining on CD47 knockout HEK-293T cells (ab266324).