Recombinant Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (ab253202)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL, 2 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5µg of ab253202 [EPR23418-23]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

Additional screenshots of mapped reads can be downloaded here.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

Blocking and diluting buffer and concentration: 5% NFDM/TBST. Fresh lysates were used in this WB.

Exposure time: Lane 1: 81 seconds; Lane 2: 3 minutes.

Immunohistochemical analysis of paraffin-embedded Human bladder cancer tissue labelling CREBBP with ab253202 at 1/2000 (0.258 µg/mL) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining in human bladder cancer (PMID: 25915404). The section was incubated with ab253202 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) was used.

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling CREBBP with ab253202 at 1/500 (1.032 µg/mL) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 µg/mL) dilution (Green). Confocal image showing mianly nuclear staining in HeLa cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 µg/mL) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 µg/mL) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling CREBBP with ab253202 at 1/500 dilution (0.1µg)/ red (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). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

CREBBP was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 µg with ab253202 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab253202 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug

Lane 2: ab253202 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab253202 in NIH/3T3 whole cell lysate

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

Exposure time: 110 seconds.

Fresh lysates were used in this IP.

Blocking and diluting buffer and concentration: 5% NFDM/TBST. Fresh lysates were used in this WB.

Exposure time: 81 seconds.

Blocking and diluting buffer and concentration: 5% NFDM/TBST. This antibody has no cross-reaction with human EP300.

These two recombinant proteins were made in-house and expressed from the E.coli expression system.

Exposure time: 1 second.

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labelling CREBBP with ab253202 at 1/2000 (0.258 µg/mL) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining in human cervical cancer. The section was incubated with ab253202 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) was used.

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

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling CREBBP with ab253202 at 1/2000 (0.258 µg/mL) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Mainly nuclear staining in mouse cerebrum. The section was incubated with ab253202 for 10 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) was used.

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

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labelling CREBBP with ab253202 at 1/2000 (0.258 µg/mL) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Mainly nuclear staining in rat cerebrum (PMID: 12445700). The section was incubated with ab253202 for 10 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) was used.

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling CREBBP with ab253202 at 1/100 (5.16 µg/mL) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 µg/mL) dilution (Green). Confocal image showing mianly nuclear staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 µg/mL) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 µg/mL) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling CREBBP with ab253202 at 1/500 dilution (0.1µg)/ red (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). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

CREBBP was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 µg with ab253202 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab253202 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.

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

Lane 2: ab253202 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab253202 in HeLa whole cell lysate

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

Exposure time: 48 seconds.

Fresh lysates were used in this IP.

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 ab253202 (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 (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers are from paper PMID:2178921

* http://www.abcam.com/resources?keywords=X%20ChIP%20protocol