Product nameCell Cycle In-Cell ELISA Kit (Fluorescent)
Sample typeAdherent cells
Assay typeCell-based (quantitative)
Assay time6h 30m
Assay durationMultiple steps standard assay
Species reactivityReacts with: Mouse, Human
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.
Tested applicationsSuitable for: In-Cell ELISAmore details
Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 1000X Anti-Mouse IgG/AP-Labeled Secondary Antibody 1 x 20µl 1000X Anti-Rabbit IgG/HRP-Labeled Secondary Antibody 1 x 20µl 100X Anti- Histone H3 (pSer10) Primary Antibody 1 x 120µl 100X Anti-Cdk2 (pTyr15) Primary Antibody 1 x 120µl 100X Triton X-100 1 x 500µl 10X Blocking Solution 1 x 10ml 10X Phosphate Buffered Saline 1 x 100ml 10X Quenching Solution 1 x 1.5ml 400X Fluorescent Substrate Cocktail 1 x 50µl 400X Tween-20 1 x 2ml 8000X Hydrogen Peroxide 1 x 50µl Fluorescent Substrate Buffer 1 x 12ml 1X Janus Green Stain 1 x 17ml
Cellular localizationCytoplasmic and Nuclear
- Cell division protein kinase 2
Our Abpromise guarantee covers the use of ab140363 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||Use at an assay dependent concentration.|
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:
Lane 1 -Untreated,
Lane 2- hydroxyurea = G1/S arrest,
Lane 3- paclitaxel = G2/M arrest .
(B) Cdk2 pTyr15 antibody:
Lane 1 -Untreated,
Lane 2-, thymidine = G1/S arrest,
Lane 3- nocodazole = G2/M arrest.
Data shown is for 24 hour treatment with 1.6 – 5000 nM hydroxyurea. Cdk2 pTyr15 intensity increases with hydroxyurea treatment dose whereas Histone H3 pSer10 intensity decreases.
Data shown is for 24 hour treatment with 1 mM hydroxyurea, 333 nM paclitaxel and untreated (Control). (Normalized intensity is described in section 9.)
This product has been referenced in:
- Tiwari KK et al. Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro. Biochem Biophys Res Commun 450:1345-50 (2014). Read more (PubMed: 24997337) »