Human Cell Cycle In-Cell ELISA kit (IR) is Cell-based (quantitative) ELISA for the measurement of Human Cell Cycle (IR) production in Mouse, Human in Suspension cells, Adherent cells samples.
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Key facts
- Detection method
- Colorimetric
- Sample types
- Suspension cells, Adherent cells
- Assay type
- Cell-based (quantitative)
- Reactive species
- Mouse, Human
Reactivity data
| Application | Reactivity | Dilution info | Notes |
|---|---|---|---|
Application ELISA | Reactivity Reacts | Dilution info - | Notes - |
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Target data
Function
Serine/threonine-protein kinase involved in the control of the cell cycle; essential for meiosis, but dispensable for mitosis (PubMed:10499802, PubMed:10884347, PubMed:10995386, PubMed:10995387, PubMed:11051553, PubMed:11113184, PubMed:12944431, PubMed:15800615, PubMed:17495531, PubMed:19966300, PubMed:20935635, PubMed:21262353, PubMed:21596315, PubMed:28216226, PubMed:28666995). Phosphorylates CABLES1, CTNNB1, CDK2AP2, ERCC6, NBN, USP37, p53/TP53, NPM1, CDK7, RB1, BRCA2, MYC, NPAT, EZH2 (PubMed:10499802, PubMed:10995386, PubMed:10995387, PubMed:11051553, PubMed:11113184, PubMed:12944431, PubMed:15800615, PubMed:19966300, PubMed:20935635, PubMed:21262353, PubMed:21596315, PubMed:28216226). Triggers duplication of centrosomes and DNA (PubMed:11051553). Acts at the G1-S transition to promote the E2F transcriptional program and the initiation of DNA synthesis, and modulates G2 progression; controls the timing of entry into mitosis/meiosis by controlling the subsequent activation of cyclin B/CDK1 by phosphorylation, and coordinates the activation of cyclin B/CDK1 at the centrosome and in the nucleus (PubMed:18372919, PubMed:19238148, PubMed:19561645). Crucial role in orchestrating a fine balance between cellular proliferation, cell death, and DNA repair in embryonic stem cells (ESCs) (PubMed:18372919, PubMed:19238148, PubMed:19561645). Activity of CDK2 is maximal during S phase and G2; activated by interaction with cyclin E during the early stages of DNA synthesis to permit G1-S transition, and subsequently activated by cyclin A2 (cyclin A1 in germ cells) during the late stages of DNA replication to drive the transition from S phase to mitosis, the G2 phase (PubMed:18372919, PubMed:19238148, PubMed:19561645). EZH2 phosphorylation promotes H3K27me3 maintenance and epigenetic gene silencing (PubMed:20935635). Cyclin E/CDK2 prevents oxidative stress-mediated Ras-induced senescence by phosphorylating MYC (PubMed:19966300). Involved in G1-S phase DNA damage checkpoint that prevents cells with damaged DNA from initiating mitosis; regulates homologous recombination-dependent repair by phosphorylating BRCA2, this phosphorylation is low in S phase when recombination is active, but increases as cells progress towards mitosis (PubMed:15800615, PubMed:20195506, PubMed:21319273). In response to DNA damage, double-strand break repair by homologous recombination a reduction of CDK2-mediated BRCA2 phosphorylation (PubMed:15800615). Involved in regulation of telomere repair by mediating phosphorylation of NBN (PubMed:28216226). Phosphorylation of RB1 disturbs its interaction with E2F1 (PubMed:10499802). NPM1 phosphorylation by cyclin E/CDK2 promotes its dissociates from unduplicated centrosomes, thus initiating centrosome duplication (PubMed:11051553). Cyclin E/CDK2-mediated phosphorylation of NPAT at G1-S transition and until prophase stimulates the NPAT-mediated activation of histone gene transcription during S phase (PubMed:10995386, PubMed:10995387). Required for vitamin D-mediated growth inhibition by being itself inactivated (PubMed:20147522). Involved in the nitric oxide- (NO) mediated signaling in a nitrosylation/activation-dependent manner (PubMed:20079829). USP37 is activated by phosphorylation and thus triggers G1-S transition (PubMed:21596315). CTNNB1 phosphorylation regulates insulin internalization (PubMed:21262353). Phosphorylates FOXP3 and negatively regulates its transcriptional activity and protein stability (By similarity). Phosphorylates ERCC6 which is essential for its chromatin remodeling activity at DNA double-strand breaks (PubMed:29203878).
Additional Targets
H3-3A
Alternative names
CDKN2, CDK2, Cyclin-dependent kinase 2, Cell division protein kinase 2, p33 protein kinase
What's included?
Recommended products
Human Cell Cycle In-Cell ELISA kit (IR) is Cell-based (quantitative) ELISA for the measurement of Human Cell Cycle (IR) production in Mouse, Human in Suspension cells, Adherent cells samples.
Key facts
- Detection method
- Colorimetric
- Sample types
- Suspension cells, Adherent cells
- Assay type
- Cell-based (quantitative)
- Reactive species
- Mouse, Human
- Assay Platform
- Microplate
Precision
Intra assay
| Sample | n | mean | SD | C.V. |
|---|---|---|---|---|
Sample Cdk2 | n 0 | mean - | SD - | C.V. 4.4 |
Sample Histone H3 | n 0 | mean - | SD - | C.V. 3.9 |
Storage
- Shipped at conditions
- Blue Ice
- Appropriate short-term storage conditions
- +4°C
- Appropriate long-term storage conditions
- +4°C
- Storage information
- +4°C
Notes
Human Cell Cycle In-Cell ELISA kit (IR) (ab139412) that uses quantitative immunocytochemistry to measure levels of Cdk2 protein phosphorylated Tyr15 and Histone H3 protein phosphorylated Ser10 levels in cultured cells. Cells are fixed in a microplate and targets of interest are detected with highly specific, well-characterized antibodies. Relative target levels are quantified using IRDye®-labeled Secondary Antibodies and IR imaging such as a LI-COR® Odyssey® or Aerius® system. Antibody signal intensity can be normalized to the total cell amount using Janus Green stain.
Plates are available in our ICE (In-Cell ELISA) Support Pack (In-Cell ELISA (ICE) Support Pack ab111542) which can be bought seperately.
The Cdk2 (pTyr15) + Histone H3 (pSer10) In-Cell ELISA Kit (IR) (ab139412) is designed to study cell cycle effects in response to various stimuli. Monoclonal antibodies specific to Cdk2 (pTyr15) and Histone H3 (pSer10) are used in this high-throughput duplexing plate-based assay. Cdk2 (pTyr15) is elevated in G1/S phase of the cell cycle and Histone H3 (pSer10) is elevated in G2/M phase. Cyclin-dependent kinase 2 (Cdk2) is a nuclear protein kinase that functions in the G1/S phase of the cell cycle. Inhibitory phosphorylation occurs on residues Thr14 and Tyr15; activation of Cdk2 includes dephosphorylation of these residues by cdc25. Cdk2 can form a complex with Cyclin A, D or E. Phosphorylation of Cdk2 at Tyr15 indicates that a cell is at the G1/S transition.
Histone H3 is one of the four core histone proteins (H2A, H2B, H3 and H4) that pack DNA in nucleosomes. Post-translational modifications of histones include phosphorylation and acetylation and are important for chromatin assembly and gene expression. Phosphorylation of Histone H3 at Ser10 is tightly correlated with chromosome condensation during mitosis. Hence, Histone H3 pSer10 signal indicates a mitotic cell with condensed DNA.
In-Cell ELISA (ICE) technology employed to perform quantitative immunocytochemistry of cultured cells with a near-infrared fluorescent dye-labeled detector antibody. The technique generates quantitative data with specificity similar to western blotting, but with much greater quantitative precision and higher throughput due to the greater dynamic range and linearity of direct fluorescence detection and the ability to run 96 samples in parallel. Because the Cdk2 (pTyr15) antibody is a rabbit antibody and the Histone H3 (pSer10) antibody is a mouse antibody, they can be measured simultaneously in the same well using the cocktail of provided primary antibodies and the provided cocktail of IRDye®-labeled species-specific secondary antibodies when using a LI-COR infrared imager. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins and their post-translational modifications, and thus eliminating changes during sample handling, such as in the preparation of protein extracts. Finally, the Cdk2 (pTyr15) and Histone H3 (pSer10) signals can be normalized to cell amount, measured by the provided Janus Green whole cell stain, to further increase the assay precision.
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Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
The biological target CdK2 also known as Cyclin-Dependent Kinase 2 and Histone H3 is involved in cell cycle regulation and chromatin remodeling. CdK2 is a serine/threonine kinase with a mass of approximately 34 kDa. It is expressed in the nucleus of dividing cells. Histone H3 is one of the core histones that form the histone octamer around which DNA is wrapped and it has a mass of about 15.3 kDa. These proteins are expressed in various tissues particularly in proliferating cells throughout the body.
- Biological function summary
Cyclin-Dependent Kinase 2 partners with specific cyclin proteins to drive cell cycle progression. It forms a complex with Cyclin E or Cyclin A to control the G1/S and S/G2 transitions ensuring proper DNA replication. Histone H3 undergoes various post-translational modifications like phosphorylation and acetylation which impact chromatin structure and gene expression. As part of the nucleosome it plays an important role in regulating access to genetic information.
- Pathways
CdK2 and Histone H3 play key roles in the cell cycle and epigenetic regulation pathways. CdK2 interacts with Cyclin E during the transition from G1 to S phase influencing DNA replication. Cyclin A is involved in the S phase and entry into mitosis. Histone H3 modifications are central to the regulation of epigenetic pathways affecting gene expression levels and stability. These pathways integrate with numerous cellular processes and interact with several proteins including RETINOBLASTOMA (Rb) and p21 which further regulate the cell cycle.
- Associated diseases and disorders
CdK2 and Histone H3 have critical roles in cancer and neurological diseases. CdK2 overactivity can lead to uncontrolled cell proliferation contributing to various cancers. Its interaction with Cyclin E is often upregulated in tumors. Aberrant modifications of Histone H3 such as mutations in H3K27 or H3K9 link to specific cancers like gliomas. Moreover dysregulation in Histone H3 is associated with Huntington's disease involving alterations in transcriptional regulation. These connections highlight the potential for targeting CdK2 and Histone H3 in therapeutic approaches.
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4 product images
In-Cell ELISA - Human Cell Cycle In-Cell ELISA kit (IR) (ab139412)
HeLa cells were treated for 24h with varying concentrations of paclitaxel (0.26 – 2000 µM). Histone H3 pSer10 (red) intensity increases with increasing paclitaxel whereas Cdk2 pTyr15 (green) intensity decreases. This is the expected result for paclitaxel treatment: mitotic arrest. (B) HeLa cells were treated for 24h with varying concentrations of hydroxyurea (0.002 – 5 mM). Histone H3 pSer10 (red) intensity decreases with increasing paclitaxel whereas Cdk2 pTyr15 (green) intensity increases. This is the expected result for hydroxyurea treatment: G1/S-phase arrest.
In-Cell ELISA - Human Cell Cycle In-Cell ELISA kit (IR) (ab139412)
Quantification of the data shown in Image 1. Data shown is for 24 hour treatment with 1 mM hydroxyurea, 333 nM paclitaxel and untreated (Control).
In-Cell ELISA - Human Cell Cycle In-Cell ELISA kit (IR) (ab139412)
Cdk2 pTyr15 intensity increases with Hydroxyurea treatment dose whereas Histone H3 pSer10 intensity decreases.
Western blot - Human Cell Cycle In-Cell ELISA kit (IR) (ab139412)
Antibody specificity demonstrated by Western Blot Analysis. Whole cell lysates from HeLa cells were analyzed by western blot with the primary antibodies used in this assay kit. (A) Histone H3 pSer10 antibody: untreated (lane 1), hydroxyurea = G1/S arrest (lane 2), paclitaxel = G2/M arrest (lane 3). (B) Cdk2 pTyr15 antibody: untreated (lane 1), thymidine = G1/S arrest (lane 2), nocodazole = G2/M arrest (lane 3).
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