Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD276 with ab219648 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous staining on dendritic cells of human tonsil is observed.

Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-CD276 antibody [EPR20115] (ab219648) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : CD276 knockout HEK293T cell lysate
Lane 3 : LNCaP cell lysate
Lane 4 : Raji 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: 57 kDa
Observed band size: 90-110 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab219648 observed at 90-110 kDa. Red - loading control ab8245 observed at 36 kDa.

ab219648 Anti-CD276 antibody [EPR20115] was shown to specifically react with CD276 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266658 (knockout cell lysate ab257097) was used. Wild-type and CD276 knockout samples were subjected to SDS-PAGE. ab219648 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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 placenta tissue labeling CD276 with ab219648 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Cytoplasmic and membranous staining on vascular endothelial cells and trophoblast cells of human placenta is observed.

Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-CD276 antibody [EPR20115] (ab219648) at 1/1000 dilution

Lane 1 : LNCaP (Human prostate cancer cell line) whole cell lysate
Lane 2 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 90-110 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Raji cells were used as a negative control for CD276 (B7-H3) (PMID: 21344157).

 Negative control: lane 2 Raji cell lysate

All lanes : Anti-CD276 antibody [EPR20115] (ab219648) at 1/2000 dilution

Lane 1 : U-2OS (Human bone osteosarcoma epithelial cell line) whole cell lysate
Lane 2 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lane 3 : 293T (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : MDA-MB-435S (Human ductal carcinoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 57 kDa
Observed band size: 90-110 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

CD276 (B7-H3) has a molecular weight of about 90–100 kDa, depending on the glycosylation levels. [PMID:22473715].

All lanes : Anti-CD276 antibody [EPR20115] (ab219648) at 1/1000 dilution

Lane 1 : Human lung lysate
Lane 2 : Human skin lysate
Lane 3 : Human placenta lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Predicted band size: 57 kDa
Observed band size: 90-110 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2: 30 seconds; Lane 3: 10 seconds.

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling CD276 with ab219648 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Negative tissue: no staining on human skeletal muscle. [PMID: 18975328].

Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma tissue labeling CD276 with ab219648 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous staining on human hepatocellular carcinoma is observed.

Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling CD276 with ab219648 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous staining on human lung carcinoma is observed [PMID: 25609202].

Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling CD276 with ab219648 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous and cytoplasmic staining on human breast cancer tumor cells and stromal cells is observed.

Counter stained with Hematoxylin.

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

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.