Description
Key facts
Detection method:Fluorescent,
Sample types:Adherent cells,
Reacts with:Mouse, Human,
Assay type:Cell-based (quantitative),
Assay time:6h 30m,
Assay Platform:Microplate
Product details:
ab140363 is an In-Cell ELISA (ICE) assay kit 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 secondary antibodies conjugated to either horseradish peroxidase (HRP) or alkaline phosphatase (AP) which generate signal through the use of two spectrally distinct fluorogenic substrates. Fluorescence is measured using a standard fluorescent spectrophotometer and relative levels of target proteins are quantified. Optionally, antibody signal intensity can be normalized to the total cell amount using Janus Green stain. In-Cell ELISA (ICE) 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 fluorescence detection and the ability to run up to 96 samples in parallel. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins and their post-translational modifications, and thus essentially eliminates changes during sample handling, such as preparation of protein extracts.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
The Cdk2 (pTyr15) + Histone H3 (pSer10) In-Cell ELISA Kit (Fluorescent) (ab140363) 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 is used to perform quantitative immunocytochemistry of cultured cells using enzyme linked secondary antibodies and fluorogenic substrates. 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 fluorescence detection and the ability to run up to 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, species-specific secondary antibodies and fluorogenic substrates. 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.
REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-+4°C, Appropriate long-term storage conditions-+4°C, Storage information-+4°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Cyclin-dependent kinase 2 (Cdk2) and Histone H3 are critical components in cell cycle regulation. Cdk2 with a mass of approximately 34 kDa is a serine/threonine kinase expressed in various tissues prominently in proliferating cells. It associates with cyclins A and E to regulate progression of the cell cycle from G1 to S phase. Histone H3 a core component of chromatin undergoes post-translational modifications that influence DNA accessibility and transcription. Together Cdk2 and Histone H3 interactions might influence cell cycle and chromatin dynamics.
Biological function summary
Histone H3 is key to chromatin structure and gene regulation. It is part of the nucleosome core which packages DNA into chromatin. Modifications like phosphorylation at serine 10 occur during mitosis and open chromatin structure. Cdk2 influences such modifications via hyperphosphorylated states contributing to DNA replication and repair processes. During these processes Cdk2 forms a complex with cyclins particularly cyclin A to ensure precise control over cell cycle transitions and genomic stability.
Pathways
Cdk2 and Histone H3 connections play roles in cell cycle and DNA damage response pathways. The cell cycle pathway with Cdk2-cyclin E/A involvement promotes G1/S phase transition. In DNA damage response Cdk2 interacts with proteins like p21 to halt cell cycle in response to DNA damage. Histone H3 post-translational changes are regulated within chromatin remodeling pathways affecting gene expression and cell cycle checkpoints integrating with Cdk2's role in the replication machinery.
Aberrations in Cdk2 and Histone H3 functioning link to cancer and neurodevelopmental diseases. Overexpression of Cdk2 can drive unregulated cell proliferation in cancers often associated with reduced function of p21 a Cdk2 inhibitor. Misregulation of Histone H3 phosphorylation correlates with chromatin defects and tumorigenesis. Interventions that target the Cdk2-Histone H3 axis might offer therapeutic potential in these disorders by correcting cycle deregulation and chromatin remodeling abnormalities.
