Anti-CREBBP antibody [EPR23418-23] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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 Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

• ChIP

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ChIP - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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

• IP

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Immunoprecipitation - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

All lanes:

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

Predicted band size: 265 kDa

Observed band size: 300 kDa

false

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

All lanes:

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

Predicted band size: 265 kDa

Observed band size: 300 kDa

false

• WB

Lab

Western blot - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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

Exposure time : 81 seconds.

All lanes:

Western blot - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (ab253202) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma), 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: 265 kDa

Observed band size: 300 kDa

false

• WB

Lab

Western blot - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

All lanes:

Western blot - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (ab253202) at 1/1000 dilution

Lane 1:

HAP1 (human chronic myelogenous leukemia near-haploid cell), whole cell lysate at 28 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 14 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: 265 kDa

Observed band size: 300 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 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 found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (AB253202)

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.

All lanes:

Western blot - Anti-CREBBP antibody [EPR23418-23] - ChIP Grade (ab253202) at 1/1000 dilution

Lane 1:

His-tagged human CREBBP recombinant protein (aa2221-2442) at 0.01 µg

Lane 2:

His-tagged human EP300 recombinant protein (aa2215-2414) at 0.01 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: 265 kDa

Observed band size: 25 kDa

false