Anti-CTCF antibody [RM1044]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CTCF antibody [RM1044] (AB300639)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling CTCF with ab300639 at 1/500 dilution (0.1µg) (red) compared with a Rabbit monoclonal IgG (ab172730) (black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [RM1044] (AB300639)

Immunohistochemical analysis of paraffin-embedded human breast tissue labelling CTCF with ab300639 at 1/10000 (0.053 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Nuclear staining on human breast is observed. The section was incubated with ab300639 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CTCF antibody [RM1044] (AB300639)

Immunofluorescent analysis of 4% paraformaldehyde fixed and 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) labeling CTCF with ab300639 at 1/2000 dilution (0.266 μg/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, green) preadsorbed at 1 : 1000 (2 μg/mL). Confocal image showing nuclear staining in HeLa cell line.

ab195889 anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (red) was used as a counterstain at 1 : 200 dilution (2.5 μg/ml). Nuclear counter satin is DAPI.

• IP

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Immunoprecipitation - Anti-CTCF antibody [RM1044] (AB300639)

CTCF was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab300639 at 1/30 dilution (2µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300639 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CTCF antibody [RM1044] (ab300639) at 1/30 dilution

All lanes:

HeLa (human cervical adenocarcinoma epithelial cell) 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

Predicted band size: 83 kDa

Observed band size: 40 kDa,130 kDa,97 kDa,80 kDa,73 kDa,55 kDa,45 kDa

false

Exposure time: 10s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [RM1044] (AB300639)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labelling CTCF with ab300639 at 1/10000 (0.053 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Nuclear staining on mouse colon. The section was incubated with ab300639 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-CTCF antibody [RM1044] (AB300639)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat colon (fresh) tissue labeling CCTF with ab300639 at 1/2000 dilution (0.266 µg/ml)  followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preabsorbed at 1/1000 dilution (2 µg/mL) (Green). Positive staining on rat colon is observed. The nuclear counterstain was DAPI (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [RM1044] (AB300639)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labelling CTCF with ab300639 at 1/10000 (0.053 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Nuclear staining on rat colon. The section was incubated with ab300639 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (BOND™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

• WB

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Western blot - Anti-CTCF antibody [RM1044] (AB300639)

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

The multiples bands observed in the WB have been reported in literature (PMID : 12878173, PMID : 8246978).

All lanes:

Western blot - Anti-CTCF antibody [RM1044] (ab300639) at 1/1000 dilution

Lane 1:

Human spleen lysate at 20 µg

Lane 2:

Mouse heart lysate at 20 µg

Lane 3:

Mouse spleen lysate at 20 µg

Lane 4:

Mouse colon lysate at 20 µg

Lane 5:

Rat heart lysate at 20 µg

Lane 6:

Rat spleen lysate at 20 µg

Lane 7:

Rat colon 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: 83 kDa

Observed band size: 140 kDa,130 kDa,97 kDa,80 kDa,73 kDa,55 kDa,45 kDa

false