Anti-CTHRC1 antibody [EPR22851-145]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTHRC1 antibody [EPR22851-145] (AB256458)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTHRC1 antibody [EPR22851-145] (AB256458)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTHRC1 antibody [EPR22851-145] (AB256458)

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.

• IP

Unknown

Immunoprecipitation - Anti-CTHRC1 antibody [EPR22851-145] (AB256458)

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.

All lanes:

Immunoprecipitation - Anti-CTHRC1 antibody [EPR22851-145] (ab256458)

Predicted band size: 26 kDa

Observed band size: 26 kDa

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• WB

Lab

Western blot - Anti-CTHRC1 antibody [EPR22851-145] (AB256458)

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.

All lanes:

Western blot - Anti-CTHRC1 antibody [EPR22851-145] (ab256458) at 1/1000 dilution

Lane 1:

SK-MEL-28 (human malignant melanoma), whole cell lysate at 20 µg

Lane 2:

SW620 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa,30 kDa

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• WB

Lab

Western blot - Anti-CTHRC1 antibody [EPR22851-145] (AB256458)

Western blot : Anti-CTHRC1 antibody [EPR22851-145] (ab256458) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab256458 was shown to bind specifically to CTHRC1. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lanes 1 - 3:

Western blot - Anti-CTHRC1 antibody [EPR22851-145] (ab256458) at 1/1000 dilution

Lane 1:

HEK-293 cell lysate at 20 µg

Lane 2:

HepG2 cell lysate at 20 µg

Lane 3:

PC-3 cell lysate at 20 µg

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