Recombinant
RabMAb

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

Overview

  • Product name

    Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade
    See all Cdk9 primary antibodies
  • Description

    Rabbit monoclonal [EPR22956-37] to Cdk9 - ChIP Grade
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IP, WB, IHC-P, ICC/IF, Flow Cyt, ChIPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Mouse Cdk9 aa 300 to the C-terminus. The exact sequence is proprietary.
    Database link: Q99J95

  • Positive control

    • 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.
  • General notes

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab239364 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP 1/30.
WB 1/1000. Detects a band of approximately 42, 55 kDa (predicted molecular weight: 43 kDa).
IHC-P 1/2000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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

ICC/IF 1/100.
Flow Cyt 1/600.
ChIP Use 5 µg for 25 µg of chromatin.

Target

  • Function

    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 production elongation by phosphorylating the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAP II), SUPT5H and RDBP. The CDK9/cyclin-K complex has also a kinase activity toward CTD of RNAP II and can substitute for P-TEFb in vitro.
  • Tissue specificity

    Ubiquitous.
  • Sequence similarities

    Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily.
    Contains 1 protein kinase domain.
  • Cellular localization

    Nucleus.
  • Information by UniProt
  • Database links

  • Alternative names

    • C-2K antibody
    • CDC2 related kinase antibody
    • CDC2L4 antibody
    • Cdk 9 antibody
    • Cdk9 antibody
    • CDK9_HUMAN antibody
    • Cell division cycle 2-like protein kinase 4 antibody
    • Cell division protein kinase 9 antibody
    • CTK1 antibody
    • Cyclin dependent kinase 9 antibody
    • Cyclin-dependent kinase 9 antibody
    • PITALRE antibody
    • Serine/threonine-protein kinase PITALRE antibody
    • TAK antibody
    • Tat associated kinase complex catalytic subunit antibody
    see all

Images

  • All lanes : 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 1-5 & 7-9 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
    Lane 6 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

    Predicted band size: 43 kDa
    Observed band size: 42,55 kDa
    why is the actual band size different from the predicted?



    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 

  • 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

     

  • 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.

  • 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

  • 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.

  • 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.

  • 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.

  • 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.

  • 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 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.

  • 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.

  • 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.

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

     

References

ab239364 has not yet been referenced specifically in any publications.

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