Anti-CD47 antibody [EPR24922-5] (BSA and Azide free)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (BSA and Azide free) (AB300436)

This data was developed using ab300435, the same antibody clone in a different buffer formulation. 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (BSA and Azide free) (AB300436)

This data was developed using ab300435, the same antibody clone in a different buffer formulation. 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 (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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (BSA and Azide free) (AB300436)

This data was developed using ab300435, the same antibody clone in a different buffer formulation. 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD47 antibody [EPR24922-5] (BSA and Azide free) (AB300436)

This data was developed using ab300435, the same antibody clone in a different buffer formulation. 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

• WB

Supplier Data

Western blot - Anti-CD47 antibody [EPR24922-5] (BSA and Azide free) (AB300436)

This data was developed using ab300435, the same antibody clone in a different buffer formulation. 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) Blocking Buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

All lanes:

Western blot - Anti-CD47 antibody [EPR24922-5] (<a href='/en-us/products/primary-antibodies/cd47-antibody-epr24922-5-ab300435'>ab300435</a>) 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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/100000 dilution

Observed band size: 47 kDa

false

• WB

Supplier Data

Western blot - Anti-CD47 antibody [EPR24922-5] (BSA and Azide free) (AB300436)

This data was developed using ab300435, the same antibody clone in a different buffer formulation. Negative control : HepG2 (PMID : 28378740) Samples are non-boiled as boiling may cause protein aggregates. Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-CD47 antibody [EPR24922-5] (<a href='/en-us/products/primary-antibodies/cd47-antibody-epr24922-5-ab300435'>ab300435</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 47 kDa

false