Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade

• ICC/IF

Unknown

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 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. 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 ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

Unknown

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 (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

Unknown

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 (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 (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• IHC-P

Unknown

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 (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 (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• ChIP

Unknown

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 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.

• ChIP-seq

Supplier Data

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.

• Flow Cyt (Intra)

Unknown

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 (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

Unknown

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 (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 (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• ICC/IF

Unknown

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 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. 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 ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

• IHC-P

Unknown

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 (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 (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• ChIP

Lab

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 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

• IP

Unknown

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) (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 (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

false

• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Lane 6:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 43 kDa

Observed band size: 42 kDa,55 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (AB239364)

CUT&RUN was performed using the 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.

• ChIC/CUT&RUN-seq

Supplier Data

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