Anti-CD47 抗体 [EPR24922-5] (ab300435)

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

Negative control: HepG2 (PMID: 28378740)

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

Exposure time: Lane 1-3: 48 seconds Lane 4: 15 seconds

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 [EPR24922-5] (ab300435) 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, ab300435 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 CRISPR-Cas9 edited cell line ab266324 (CRISPR-Cas9 edited cell lysate ab257220). The band observed in the CRISPR-Cas9 edited lysate lane above 90kDa is likely to represent CD47 with an insertion. The band below 47 kDa in the CRISPR-Cas9 edited lysate lane is likely to represent a truncated form of CD47. This has not been investigated further and the functional properties of the gene products have not been determined. To generate this image, wild-type and CD47 CRISPR-Cas9 edited 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)

Immunohistochemical analysis of paraffin-embedded human wild-type and CD47 CRISPR-Cas9 edited cells labeling CD47 with ab300435 at 1/500 dilution (0.098 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on (A) Human wild-type HEK-293T cells and almost no staining on (B) Human CD47 CRISPR-Cas9 edited HEK-293T cells (ab266324). The section was incubated with ab300435 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling CD47 with ab300435 at 1/2000 dilution (0.244 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on human gastric cancer (PMID: 28693236). The section was incubated with ab300435 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human ovarian cancer tissue labeling CD47 with ab300435 at 1/2000 dilution (0.244 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on human ovarian cancer (PMID: 28380460). The section was incubated with ab300435 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD47 with ab300435 at 1/2000 dilution (0.244 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection kit). Positive staining on human tonsil. The section was incubated with ab300435 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: PBS was used instead of primary antibody, followed by a ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.