Recombinant 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).

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).

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. 

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. 

Immunohistochemical analysis of paraffin-embedded human breast carcinoma 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 carcinoma 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).

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

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).

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

Immunohistochemical analysis of paraffin-embedded human 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 human colon 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).

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

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse 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 mouse colon is observed. The nuclear counterstain was DAPI (Blue).

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).

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. 

Immunofluorescent analysis of 4% paraformaldehyde fixed and 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells 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 NIH/3T3 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. 

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.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) 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.

CTCF was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysat 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.

Lane 1: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate 10 μg (Input)

Lane 2: ab300639 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300639 in HeLa whole cell lysate

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

Exposure time: 10 seconds. 

Observed MW (kDa): 40, 130, 97, 80, 73, 55, 45. 

Chromatin was prepared from HeLa cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab300639(red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol.