All lanes : Anti-CD276 antibody [SP206] (ab227670) at 1/400 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CD276 knockout HeLa cell lysate
Lane 3 : LNCaP cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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 - ab227670 observed at 90-110 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab227670 was shown to react with CD276 antigen in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261756 (knockout cell lysate ab257098) was used. Wild-type HeLa and CD276 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab227670 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 400 dilution and a 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling CD276 with ab227670 at 1/100 dilution (2.29 µg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Mainly membranous staining on the human lung carcinoma, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab227670 for 10 mins at room temperature.

All lanes : Anti-CD276 antibody [SP206] (ab227670) 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 - ab227670 observed at 90-110 kDa. Red - loading control ab8245 observed at 36 kDa.

ab227670 Anti-CD276 antibody [SP206] 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. ab227670 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.

 

Anti-CD276 antibody [SP206] (ab227670) at 1/400 dilution + HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate

Predicted band size: 57 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast tissue sections labeling CD276 with ab227670 at 1/100 dilution (2.29 µg/ml). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Mainly membranous staining on the human breast, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab227670 for 10 mins at room temperature.

Formalin-fixed, paraffin-embedded human lung SCCa tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human tonsil tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human testis tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human stomach adenocarcinoma tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human lung tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human lung adenocarcinoma tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human colon tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human colon adenocarcinoma tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human breast tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human breast ductal carcinoma tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human stomach tissue stained for CD276 with ab227670 at 1/100 dilution in immunohistochemical analysis.