Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)

Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364) is a rabbit monoclonal antibody that is used to detect Cdk9 in Western Blot, Flow Cytometry (Intra), Flow Cytometry, IP, IHC-P, ICC/IF, ChIP. Suitable for Human, Mouse, Rat samples.

- Using biophysical QC, antibody identity is confirmed at a molecular level for unrivalled batch-batch consistency

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ChIP - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (AB239364), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ChIC/CUT&RUN-seq	IP	ChIP	WB	ICC/IF	ChIP-seq	Flow Cyt (Intra)	IHC-P
Human	Tested	Expected	Tested	Tested	Tested	Tested	Tested	Tested
Mouse	Expected	Tested	Tested	Tested	Tested	Expected	Tested	Tested
Rat	Expected	Expected	Expected	Tested	Expected	Expected	Expected	Tested

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 5 µg	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/30	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Human, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 5 µg for 25.00000 µg chromatin	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 5 µg for 25.00000 µg chromatin	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/1000	Notes -
Species Rat	Dilution info 1/1000	Notes -
Species Human	Dilution info 1/1000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/100	Notes -
Species Human	Dilution info 1/100	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 8 µg for 10⁷ Cells	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/600	Notes -
Species Human	Dilution info 1/600	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/2000	Notes Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/2000	Notes Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/2000	Notes Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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Target data

See full target information CDK9

Function

Protein kinase involved in the regulation of transcription (PubMed:10574912, PubMed:10757782, PubMed:11145967, PubMed:11575923, PubMed:11809800, PubMed:11884399, PubMed:14701750, PubMed:16109376, PubMed:16109377, PubMed:20930849, PubMed:28426094, PubMed:29335245). Member of the cyclin-dependent kinase pair (CDK9/cyclin-T) complex, also called positive transcription elongation factor b (P-TEFb), which facilitates the transition from abortive to productive elongation by phosphorylating the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAP II) POLR2A, SUPT5H and RDBP (PubMed:10574912, PubMed:10757782, PubMed:11145967, PubMed:11575923, PubMed:11809800, PubMed:11884399, PubMed:14701750, PubMed:16109376, PubMed:16109377, PubMed:20930849, PubMed:28426094, PubMed:30134174). This complex is inactive when in the 7SK snRNP complex form (PubMed:10574912, PubMed:10757782, PubMed:11145967, PubMed:11575923, PubMed:11809800, PubMed:11884399, PubMed:14701750, PubMed:16109376, PubMed:16109377, PubMed:20930849, PubMed:28426094). Phosphorylates EP300, MYOD1, RPB1/POLR2A and AR and the negative elongation factors DSIF and NELFE (PubMed:10912001, PubMed:11112772, PubMed:12037670, PubMed:20081228, PubMed:20980437, PubMed:21127351, PubMed:9857195). Regulates cytokine inducible transcription networks by facilitating promoter recognition of target transcription factors (e.g. TNF-inducible RELA/p65 activation and IL-6-inducible STAT3 signaling) (PubMed:17956865, PubMed:18362169). Promotes RNA synthesis in genetic programs for cell growth, differentiation and viral pathogenesis (PubMed:10393184, PubMed:11112772). P-TEFb is also involved in cotranscriptional histone modification, mRNA processing and mRNA export (PubMed:15564463, PubMed:19575011, PubMed:19844166). Modulates a complex network of chromatin modifications including histone H2B monoubiquitination (H2Bub1), H3 lysine 4 trimethylation (H3K4me3) and H3K36me3; integrates phosphorylation during transcription with chromatin modifications to control co-transcriptional histone mRNA processing (PubMed:15564463, PubMed:19575011, PubMed:19844166). The CDK9/cyclin-K complex has also a kinase activity towards CTD of RNAP II and can substitute for CDK9/cyclin-T P-TEFb in vitro (PubMed:21127351). Replication stress response protein; the CDK9/cyclin-K complex is required for genome integrity maintenance, by promoting cell cycle recovery from replication arrest and limiting single-stranded DNA amount in response to replication stress, thus reducing the breakdown of stalled replication forks and avoiding DNA damage (PubMed:20493174). In addition, probable function in DNA repair of isoform 2 via interaction with KU70/XRCC6 (PubMed:20493174). Promotes cardiac myocyte enlargement (PubMed:20081228). RPB1/POLR2A phosphorylation on 'Ser-2' in CTD activates transcription (PubMed:21127351). AR phosphorylation modulates AR transcription factor promoter selectivity and cell growth. DSIF and NELF phosphorylation promotes transcription by inhibiting their negative effect (PubMed:10912001, PubMed:11112772, PubMed:9857195). The phosphorylation of MYOD1 enhances its transcriptional activity and thus promotes muscle differentiation (PubMed:12037670). Catalyzes phosphorylation of KAT5, promoting KAT5 recruitment to chromatin and histone acetyltransferase activity (PubMed:29335245).

Alternative names

CDC2L4, TAK, CDK9, Cyclin-dependent kinase 9, C-2K, Cell division cycle 2-like protein kinase 4, Cell division protein kinase 9, Serine/threonine-protein kinase PITALRE, Tat-associated kinase complex catalytic subunit

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR22956-37
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

What is this antibody validated in?

Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364) is a rabbit recombinant monoclonal antibody and is validated for use in Western Blot (WB), Flow Cytometry (Intra), Flow Cytometry (Flow Cyt), Immunoprecipitation (IP), Immunohistochemistry (IHC-P), Immunocytochemistry/immunofluorescence (ICC/IF), ChIP, in Human, Mouse, Rat samples.

What is the molecular weight of Cdk9?

Anti-Cdk9 [EPR22956-37] - ChIP Grade (ab239364) specifically detects a band for Cdk9 (UniProt: Q99J95) at a molecular weight of 43kDa.

Recommended positive controls

WB: HEK-293T, HeLa, RAW 264.7, PC-12, NIH/3T3 and C6 whole cell lysate; Mouse brain and rat lung tissue lysate; IHC-P: Human pancreas, pancreatic cancer, mouse and rat liver tissue.ICC/IF: HeLa and NIH/3T3 cells.Flow: HeLa and NIH/3T3 cells.IP: NIH/3T3 cell lysate.ChIP: Chromatin from MEF and HeLa cells.ChIC/CUT&RUN-Seq: HeLa cells.

Trial sizes available!

Test your antibody or perform pre-screening before committing to a larger quantity. Sold in 10µl. Discover our selection of trial-size antibodies.Other related products

We have a range of other formats of antibody clone [EPR22956-37] also available for your convenience:
ab239364, Carrier free - ab259268

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine protein kinase involved in regulating transcription elongation. It is often referred to as part of the positive transcription elongation factor b (P-TEFb) complex. Cdk9 partners with cyclin T1 T2 or K to form this complex allowing it to phosphorylate the carboxyl-terminal domain (CTD) of RNA polymerase II. The molecular weight of Cdk9 is around 42 kDa. This protein is ubiquitously expressed found in many tissues including the heart liver and brain reflecting its important role.

Biological function summary

Cdk9 functions as a central component of transcriptional processes. It is essential for the transition of RNA polymerase II from the initiation phase to productive elongation. As part of the P-TEFb complex Cdk9 targets negative elongation factors helping relieve their inhibition. This regulation is especially important in the control of genes with strong promoter proximally paused polymerase. By doing so Cdk9 controls the expression of several genes involved in cell growth differentiation and response to stress.

Pathways

Cdk9 plays a significant role in transcription and signal transduction pathways. One important pathway is the NF-κB signaling where Cdk9 regulates the transcription of NF-κB target genes involved in immune response. Another significant pathway is the heat shock response where Cdk9 enhances the expression of heat shock proteins helping cells deal with stress. In both pathways it cooperates with other proteins like cyclins and negative elongation factors illustrating its interconnected role in cellular function.

Associated diseases and disorders

Cdk9 has been linked to several conditions most notably cancer and cardiac hypertrophy. Its overactivation can lead to increased transcription of genes involved in proliferation making it a focus for cancer research. Cdk9 is also implicated in cardiac hypertrophy where its activity affects gene expression that can exacerbate the condition. Researchers study its interaction with other proteins such as THZ1 a Cdk9 inhibitor to explore potential therapeutic avenues.

Product promise

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15 product images

ChIP - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Chromatin was prepared from MEF 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 ab239364 (red), or 5 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 20 μl of Protein A/G Sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are from paper PMC4103662 (PMID: 23663783)
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol
ChIC/CUT&RUN sequencing - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ChIC/CUT&RUN pAG-MNase ab285373, 105 HeLa cells and 5μg of ab239364 [EPR22956-37]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 4 μg of ab239364 [EPR22956-37]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here.
Immunoprecipitation - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
CDK9 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug with ab239364 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab239364 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
Lane 1: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug
Lane 2: ab239364 IP in NIH/3T3 whole cell lysate
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab239364 in NIH/3T3 whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 6 seconds
All lanes: Immunoprecipitation - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Predicted band size: 43 kDa
Observed band size: 42 kDa, 55 kDa
ChIP-sequencing - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 8 μg of ab239364 [EPR22956-37]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here.
Western blot - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Blocking and Dilution Buffer and concentration: 5% NFDM/TBST
Exposure times.
Lanes 1-3: 36 seconds Lanes 4-5: 5.5 seconds Lane 6: 3 minutes Lanes 7-8: 15 seconds Lane 9: 48 seconds
All lanes: Western blot - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364) at 1/1000 dilution
Lane 1: 293T (human embryonic kidney epithelial cell), whole cell lysate at 10 µg
Lane 2: HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 10 µg
Lane 3: Mouse lung tissue lysate at 20 µg
Lane 4: RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 10 µg
Lane 5: Mouse brain tissue lysate 20 ug
Lane 6: Rat lung tissue lysate at 20 µg
Lane 7: PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg
Lane 8: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg
Lane 9: C6 (rat glial tumor glial cell) whole cell lysate at 10 µg
Secondary
Lanes 1, 2, 3, 4, 5, 7, 8 and 9: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Lane 6: Western blot - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution
Predicted band size: 43 kDa
Observed band size: 42 kDa, 55 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on rat liver (PMID: 9766517, 11282025). The section was incubated with ab239364 for 10 mins at RT. 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 Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
ChIP - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
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 ab239364 (red), or 5 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 20 μl of Protein A/G Sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are from paper PMCID: PMC2756882.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on mouse liver (PMID: 9766517, 11282025) The section was incubated with ab239364 for 10 mins at RT. 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 Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Immunohistochemical analysis of paraffin-embedded Human pancreatic cancer tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on human pancreatic cancer (PMID: 28231737) The section was incubated with ab239364 for 10 mins at RT. 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 Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Nuclear staining on human pancreas (PMID: 28231737) The section was incubated with ab239364 for 10 mins at RT. 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 Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
Immunocytochemistry/ Immunofluorescence - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling CDK9 with ab239364 at 1/100 (6 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cell line is observed. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab239364 anti-CDK9 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.
Immunocytochemistry/ Immunofluorescence - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling CDK9 with ab239364 at 1/100 (6 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in HeLa cell line is observed. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab239364 anti-CDK9 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.
Flow Cytometry (Intracellular) - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma) cells labelling CDK9 with ab239364 at 1/600 (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
Flow Cytometry (Intracellular) - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryo) cells labelling CDK9 with ab239364 at 1/600 (Red) compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
ChIC/CUT&RUN sequencing - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364)
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 5 µg of ab239364 [EPR22956-37]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.

The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 8 µg of ab239364. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.

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