All lanes : Anti-Eph receptor B4/HTK antibody [EPR23222-24] (ab254301) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : EPHB4 knockout HEK293T cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : HT-29 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: 108 kDa
Observed band size: 125 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab254301 observed at 125 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab254301 Anti-Eph receptor B4/HTK antibody [EPR23222-24] was shown to specifically react with Eph receptor B4/HTK in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266733 (knockout cell lysate ab257429) was used. Wild-type and Eph receptor B4/HTK knockout samples were subjected to SDS-PAGE. ab254301 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 breast cancer tissue labeling Eph receptor B4 with ab254301 at 1/1000 dilution (0.545ug/ml) followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution. Mainly membranous staining in cancer cells of human breast cancer (PMID: 16816380) is observed. The section was incubated with ab255611 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

All lanes : Anti-Eph receptor B4/HTK antibody [EPR23222-24] (ab254301) at 1/1000 dilution

Lane 1 : GR-M (mouse Caucasian melanoma epithelial cell), whole cell lysate
Lane 2 : 4T1 (mouse mammary gland carcinoma epithelial cell), whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : MGMT overexpression 293T lysate (whole cell) (ab94051) at 1/100000 dilution

Predicted band size: 108 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 92 seconds.

The expression profile observed is consistent with what has been described in the literature (PMID: 19366806, 16840724).

All lanes : Anti-Eph receptor B4/HTK antibody [EPR23222-24] (ab254301) at 1/1000 dilution

Lane 1 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : HUVEC (human umbilical vein endothelial cell), whole cell lysate
Lane 3 : SW620 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 5 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 6 : T47D (human ductal breast epithelial tumor epithelial cell), whole cell lysate
Lane 7 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate
Lane 8 : NIH:OVCAR-3 (human ovary adenocarcinoma epithelial cell), whole cell lysate
Lane 9 : SK-BR-3 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 108 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 70 seconds.

Negative control: SW620 (PMID: 19366806).

EphB4 is negative in SW620 and positive in HT29 and HUVEC, which is different from the expression profiles of other EphB family members. The expression profile observed is consistent with what has been described in the literature (PMID: 19366806, 16840724).

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labeling Eph receptor B4 with ab254301 at 1/1000 dilution (0.545ug/ml) followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at a ready to use dilution. Positive staining in cancer cells of human colon cancer (PMID: 19366806) is observed. The section was incubated with ab255611 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Eph receptor B4 with ab254301 at 1/1000 dilution (0.545ug/ml) followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at a ready to use dilution. Weak positive staining in base of crypts of human colon (PMID:19366806) is observed. The section was incubated with ab255611 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.