Human Cell Cycle In-Cell ELISA kit (IR) (ab139412)

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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