All lanes : Anti-CTHRC1 antibody [EPR22851-145] (ab256458) at 1/1000 dilution

Lane 1 : SK-MEL-28 (human malignant melanoma), whole cell lysate
Lane 2 : SW620 (human colorectal 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: 26 kDa
Observed band size: 26,30 kDa why is the actual band size different from the predicted?

30KDa and 26KDa respectively represent glycosylated and non-glycosylated form. There are three isoforms of CTHRC1. The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 26918341). 

Negative control: SW620 (PMID: 22321245).

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

Exposure Time: 10 seconds.

CTHRC1 was immunoprecipitated from 0.35 mg SK-MEL-28 (Human malignant melanoma) whole cell lysate with ab256458 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256458 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: SK-MEL-28 (Human malignant melanoma) whole cell lysate 10ug

Lane 2: ab256458 IP in SK-MEL-28 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256458 in SK-MEL-28 whole cell lysate.

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

Exposure time: 30 seconds.

 

Immunohistochemical analysis of paraffin-embedded Human lung and lung carcinoma tissue labeling CTHRC1 with ab256458 at 1/500 dilution (1.19 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). No staining on the paracancerous human lung tissue (panel A). Positive staining on human lung carcinoma (panel B; PMID: 29631554). The section was incubated with ab256458 for 15 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 carcinoma tissue labeling CTHRC1 with ab256458 at 1/500 dilution (1.19 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the stromal cells in the human colon carcinoma, nearly no staining on the paracancerous colon tissues (PMID: 24945147). The section was incubated with ab256458 for 15 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 breast and breast carcinoma tissue labeling CTHRC1 with ab256458 at 1/500 dilution (1.19 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). No staining on the paracancerous human breast tissue (panel A). Positive staining on the stromal cells in the human breast carcinoma (panel B; PMID: 24945147). The section was incubated with ab256458 for 15 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.