Anti-CD47 antibody [EPR24922-5]

6 product images

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (ab300435)

  Immunohistochemical analysis of paraffin-embedded (A) Human wild-type tissue labeling CD47 with ab300435 at 1/5000 (0.098 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on (A) Human wild-type HEK-293T cells and almost no staining on (B) Human CD47 CRISPR-Cas9 edited HEK-293T cells (Human CD47 knockout HEK-293T cell line 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: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (ab300435)

  Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labeling CD47 with ab300435 at 1/2000 (0.244 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (ab300435)

  Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling CD47 with ab300435 at 1/2000 (0.244 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human ovarain 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: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (ab300435)

  Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling CD47 with ab300435 at 1/2000 (0.244 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. 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: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.
  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• 

  Western blot - Anti-CD47 antibody [EPR24922-5] (ab300435)

  Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBSLysates/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] (Anti-GAPDH antibody [6C5] - Loading Control 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 Human CD47 knockout HEK-293T cell line ab266324 (CRISPR-Cas9 edited cell lysate Human CD47 knockout HEK-293T 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution. Negative control: HepG2 (PMID: 28378740)

  All lanes: Western blot - Anti-CD47 antibody [EPR24922-5] (ab300435) at 1/1000 dilution

  Lanes 1 - 3: IMR-32 (human neuroblastoma neuroblast) whole cell lysate

  Lane 4: HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/100000 dilution

  Observed band size: 47 kDa

• 

  Western blot - Anti-CD47 antibody [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.

  All lanes: Western blot - Anti-CD47 antibody [EPR24922-5] (ab300435) at 1/1000 dilution

  Lane 1: Jurkat (human T cell leukemia T lymphocyte) whole cell lysate

  Lane 2: HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate 20

  Lane 3: U937 (human histiocytic lymphoma monocyte) whole cell lysate

  Lane 4: NIH:OVCAR-3 (human ovary adenocarcinoma epithelial cell) whole cell lysate

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 47 kDa