All lanes : Anti-CD105 antibody [EPR19911-220] (ab252345) at 1 µg/ml

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : ENG knockout HeLa whole cell lysate
Lane 3 : HUVEC whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 71 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab252345 observed at 95 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab252345 was shown to recognize ENG (Endoglin) in wild-type HeLa cells as signal was lost at the expected MW in ENG knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and ENG knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab252345 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling CD105 with ab252345 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Positive staining on peritubular microvasculature of rat kidney (PMID: 25381426) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use.

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

The section was incubated with ab252345 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling CD105 with ab252345 at 1/2000 dilution, followed by the Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Positive staining on sinusoidal endothelial cells of human liver (PMID: 30563158) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use.

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

The section was incubated with ab252345 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling CD105 with ab252345 at 1/2000 dilution, followed by the Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use. Positive staining on glomerular and peritubular microvasculature of human kidney (PMID: 25381426) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) ready to use.

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

The section was incubated with ab252345 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

 

CD105 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab252345 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab252345 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg (Input).
Lane 2: ab252345 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252345 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

All lanes : Anti-CD105 antibody [EPR19911-220] (ab252345) at 1/1000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HUVEC (human umbilical vein endothelial cell line) whole cell lysate
Lane 3 : Rat spleen 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: 71 kDa
Observed band size: 190,95 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 26 seconds.

Lane 2: 6 seconds.

Lane 3: 3 minutes.

The expression profile / molecular weight observed is consistent with what has been described in the literature (PMID: 12746487; 9872992).