Anti-CD105 antibody [EPR22811-18] (ab231774) at 1/1000 dilution + U937 (human histiocytic lymphoma monocyte), whole cell lysate 40 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 70 kDa
Observed band size: 97 kDa why is the actual band size different from the predicted?


Exposure time: 103 seconds

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID: 8932339).

All lanes : Anti-CD105 antibody [EPR22811-18] (ab231774) at 1/1000 dilution

Lane 1 : HUVEC (human umbilical vein endothelial cell), whole cell lysate 20 µg
Lane 2 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate 20 µg
Lane 3 : Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate 20 µg
Lane 4 : Human lung tissue lysate 20 µg
Lane 5 : Human placenta tissue lysate 20 µg

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 4-5 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 70 kDa

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

Exposure times: Lanes 1-3: 26 secs; Lanes 4-5: 3 mins.

The molecular weight observed is consistent with what has been described in the literature (PMID: 8932339).

This blot was developed using a higher sensitivity ECL substrate.

Negative control: Jurkat (PMID: 28351936); Raji (PMID: 28351936).

CD105 was immunoprecipitated from 0.35 mg HUVEC (human umbilical vein endothelial cell) whole cell lysate with ab231774 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab231774 1/1000 dilution (0.51 μg/ml). VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: HUVEC (human umbilical vein endothelial cell) whole cell lysate 10μg
Lane 2: ab231774 IP in HUVEC whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab231774 in HUVEC whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 mins.

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma tissue labeling CD105 with ab231774 at 1/100 dilution (5.1 μg/ml)  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on endothelial cells of human ovarian carcinoma (PMID: 17502949). The section was incubated with ab231774 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

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

Immunohistochemical analysis of paraffin-embedded human placenta tissue labeling CD105 with ab231774 at 1/100 dilution (5.1 μg/ml)  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human placental trophoblasts (PMID: 17956952) is observed. The section was incubated with ab231774 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

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

Immunofluorescent analysis of 100% methanol-fixed U-937 (human histiocytic lymphoma monocyte) cells labeling CD105 with ab231774 at 1/500 dilution, followed by a ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in U-937 cells is observed. ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)  was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Negative control: Jurkat (PMID: 28351936).

Flow cytometric analysis of U-937 (human histiocytic lymphoma monocyte) cells labeling CD105 with ab231774 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells. 

Flow cytometric analysis of Jurkat (human T cell leukemia T lymphocyte, Left) / HUVEC (human umbilical vein endothelial cell, Right) cells labeling CD105 with ab231774 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: Jurkat (PMID: 28351936). Gated on viable cells.