Anti-CD47 antibody [EPR24922-21]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD47 antibody [EPR24922-21] (AB300124)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Jurkat (Human T cell leukemia T lymphocyte) cells labelling CD47 with ab300124 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) antibody at 1/1000 dilution.

Confocal image showing membranous staining in Jurkat cell line.

Negative control : HepG2 (PMID : 28378740).

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

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

• Flow Cyt

Lab

Flow Cytometry - Anti-CD47 antibody [EPR24922-21] (AB300124)

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 compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Gated on viable cells.

Positive staining on HEK-293T cells (ab255449), while no staining on CD47 knockout HEK-293T cells (ab266324).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD47 antibody [EPR24922-21] (AB300124)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CD47 KO HEK293T (CD47 knockout human embryonic kidney epithelial cell) cells labelling CD47 with ab300124 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) antibody at 1/1000 dilution.

Confocal image showing membranous staining in Parental HEK293T cell line and no staining in CD47 KO HEK293T cell line.

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

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

• WB

Lab

Western blot - Anti-CD47 antibody [EPR24922-21] (AB300124)

Exposure time : Lane 1 : 10 seconds; Lane 2 : 180 seconds

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining.

Negative control : HepG2 (PMID : 28378740).

Samples are non-boiled as boiling may cause protein aggregates.

All lanes:

Western blot - Anti-CD47 antibody [EPR24922-21] (ab300124) at 1/1000 dilution

Lane 1:

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

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

U937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 4:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

NIH:OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

Lanes 1 - 5:

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 1/100000 dilution

Lanes 1 - 5:

Western blot - Anti-GAPDH antibody [EPR16891] - Loading Control (<a href='/en-us/products/primary-antibodies/gapdh-antibody-epr16891-loading-control-ab181602'>ab181602</a>) at 1/100000 dilution

Observed band size: 47-52 kDa

false

• WB

Lab

Western blot - Anti-CD47 antibody [EPR24922-21] (AB300124)

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).

All lanes:

Western blot - Anti-CD47 antibody [EPR24922-21] (ab300124) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

CD47 knockout HEK-293T whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Observed band size: 47-52 kDa

false