Anti-CCK4 antibody [EPR30150-507]

• Flow Cyt

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Flow Cytometry - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Flow cytometric analysis of A549 (human lung carcinoma epithelial cell, Left) / A431 (human epidermoid carcinoma epithelial cell, Right) cells labelling CCK4 with ab325170 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Low expression : A549.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded (A) A431 (human epidermoid carcinoma epithelial cell) cell pellet (B) HCT116 (human colorectal carcinoma epithelial cell) cell pellet (C) HT-1080 (human fibrosarcoma epithelial cell) cell pellet (D) A549 (human lung carcinoma epithelial cell) cell pellet tissue labeling CCK4 with ab325170 at 1/2000 (0.257 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on A431 (image A) and HCT116 (image B), no staining on HT-1080 (image C) and A549 (image D). The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labeling CCK4 with ab325170 at 1/500 (1.026 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human lung carcinoma. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling CCK4 with ab325170 at 1/500 (1.026 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon carcinoma. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling CCK4 with ab325170 at 1/500 (1.026 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast carcinoma. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling CCK4 with ab325170 at 1/500 (1.026 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A431 (human epidermoid carcinoma epithelial cell); A549 (human lung carcinoma epithelial cell) cells labelling CCK4 with ab325170 at 1/200 (2.565 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing membrane staining in A431 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Low expression : A549.

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab325170 at 1/200 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded Human ovarian carcinoma tissue labeling CCK4 with ab325170 at 1/500 (1.026 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human ovarian carcinoma. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-CCK4 antibody [EPR30150-507] (AB325170)

CCK4 was immunoprecipitated from 0.35 mg HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate with ab325170 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325170 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CCK4 antibody [EPR30150-507] (ab325170) at 1/30 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate 10 μg

Lane 2:

ab325170 1:30 IP in HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325170 in HCT 116 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 10s

• IP

Supplier Data

Immunoprecipitation - Anti-CCK4 antibody [EPR30150-507] (AB325170)

CCK4 was immunoprecipitated from 0.35 mg A431 (human epidermoid carcinoma epithelial cell) whole cell lysate with ab325170 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325170 at 1/1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CCK4 antibody [EPR30150-507] (ab325170) at 1/30 dilution

Lane 1:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate

Lane 2:

ab325170 1:30 IP in A431 (human epidermoid carcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325170 in A431 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3s

• WB

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Western blot - Anti-CCK4 antibody [EPR30150-507] (AB325170)

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

Low expression : A549, HT-1080(PMID : 25006253), colon

The expression profile observed is consistent with what has been described in the literature(PMID : 25006253)

This antibody can recognize both the full-length PTK7 and the N-terminal PTK7. The 160 kDa form may represent the full-length protein with glycosylation modifications, while the 110 kDa form likely corresponds to the N-terminal PTK7 glycosylation modification.

The identity of the bands lower than 37kDa are unknown.

Lanes 1-6 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 and lanes 7-9 are incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-CCK4 antibody [EPR30150-507] (ab325170) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

Human lung tissue lysate at 20 µg

Lane 8:

Human colon tissue lysate at 20 µg

Secondary

Lanes 1 - 6:

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 1/20000 dilution

Lanes 7 - 8:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 110 kDa,160 kDa,36 kDa

false

Exposure time: 180s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling CCK4 with ab325170 at 1/500 (1.026 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on human cardiac muscle. The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CCK4 antibody [EPR30150-507] (AB325170)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling CCK4 with ab325170 at 1/500 (1.026 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on human colon (PMID : 27499181). The primary antibody was incubated 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins