Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling CD47 with ab218810 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous staining on human placenta (PMID: 7998989). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

All lanes : Anti-CD47 antibody [EPR21794] (ab218810) at 1/500 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : CD47 knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 35 kDa
Observed band size: 47-52 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab218810 observed at 47-52 kDa. Red - loading control ab8245 observed at 36 kDa.

ab218810 Anti-CD47 antibody [EPR21794] was shown to specifically react with CD47 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266324 (knockout cell lysate ab257220) was used. Wild-type and CD47 knockout samples were subjected to SDS-PAGE. ab218810 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling CD47 with ab218810 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weak cytoplasmic staining on sinus of human liver sinusoidal endothelial cells (PMID: 7998989). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Immunohistochemical analysis of paraffin-embedded human ovary carcinoma tissue labeling CD47 with ab218810 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Membranous and weakly cytoplasmic staining in human ovary carcinoma (PMID: 7998989). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use. 

Lanes 1-4 : Anti-CD47 antibody [EPR21794] (ab218810) at 1/5000 dilution
Lane 5 : Anti-CD47 antibody [EPR21794] (ab218810) at 1/1000 dilution

Lane 1 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg
Lane 2 : U937 (human histiocytic lymphoma cell line) whole cell lysate at 20 µg
Lane 3 : A549 (human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4 : U-2 OS (human bone osteosarcoma epithelial cell line) whole cell lysate at 20 µg
Lane 5 : Human brain lysate at 10 µg

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

Predicted band size: 35 kDa
Observed band size: 47-52 kDa why is the actual band size different from the predicted?

 

Exposure time: Lane 1: 26 seconds; Lane 2: 70 seconds; Lanes 3-4: 3 minutes; Lane 5: 3 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

We recommend that samples are not boiled after adding loading buffer as this may cause protein aggregates. The molecular mass observed is consistent with the literature (PMID 12393467, PMID 11034562).