Anti-CDCP1 antibody [EPR23162-102] (ab252947) at 1/1000 dilution + Human colon cancer lysate at 20 µg

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 92 kDa
Observed band size: 135,70 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

The expression profile observed is consistent with what has been described in the literature (PMID: 20551327). Post-translational processing generates a C-terminal 70-kDa fragment from the full-length 135 kDa CDCP1.

All lanes : Anti-CDCP1 antibody [EPR23162-102] (ab252947) at 1/1000 dilution

Lane 1 : PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : MCF7 (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: 92 kDa
Observed band size: 135,70 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1:48 seconds;
Lanes 2-3:3 minutes.

The expression profile observed is consistent with what has been described in the literature (PMID: 20551327). Post-translational processing generates a C-terminal 70-kDa fragment from the full-length 135 kDa CDCP1.

Negative control: MCF7 (PMID: 20551327, 24055141).

CDCP1 was immunoprecipitated from 0.35 mg PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate with ab252947 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab252947 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab252947 IP in PC-3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252947 in PC-3 whole cell lysate

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

Exposure time: 3 min

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized MCF7 (Human breast adenocarcinoma epithelial cell, Left) / HCT 116 (Human colorectal carcinoma epithelial cell, Right) cells labeling CDCP1 with ab252947 at 1/500 dilution (Red) compared with a Isotype control details (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Secondary antibody details (ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control: MCF7 cell line (PMID: 24055141).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HCT 116 (Human colorectal carcinoma epithelial cell) MCF7 (Human breast adenocarcinoma epithelial cell) cells labelling CDCP1 with ab252947 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing membranous staining in HCT 116 cell line. Negative control: MCF7 cell line (PMID: 24055141). 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: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000  dilution.

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling CDCP1 with ab252947 at 1/200 dilution (2.8 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly membranous staining in tumor cells of human breast cancer (PMID: 16823897). The section was incubated with ab252947 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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 cancer tissue labeling CDCP1 with ab252947 at 1/200 dilution (2.8 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly membranous staining in tumor cells of human colon cancer (PMID: 12660814). The section was incubated with ab252947 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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 CDCP1 with ab252947 at 1/200 dilution (2.8 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining in human colon (PMID: 12660814). The section was incubated with ab252947 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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