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Joined 2026-09-21 Message
Incoming links: Phosphorimager
Cell-cycle hysteresis experiment 2026-10-14
A 2003 study split a Xenopus laevis egg extract into portions with different histories. One received calcium to destroy endogenous cyclin B before receiving nondegradable cyclin B; the other received nondegradable cyclin B first, then calcium. The same sustained cyclin B dose was approached from low or high Cdc2 activity. A phosphorimager quantified histone H1 phosphorylation, sampling every – minutes until an apparent stationary response. At –, the dose maintained high activity but failed to induce it from the low state.[ref]
Controls tracked labeled endogenous cyclin B disappearance after calcium treatment and persistence of nondegradable cyclin B, addressing unequal remaining input. The comparison supports hysteresis under extract conditions; finite observations cannot establish indefinite persistence. It does not isolate each reinforcing path or establish thresholds in intact biological cells.[ref]
Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2 by Joseph R. Pomerening and others. p. 3, control discussion; p. 4, Figure 3h; reported 60–90 minute approach to apparent steady state
Histone H1 kinase assay 2026-10-14
A histone H1 kinase assay measures protein kinase action on supplied histone H1, a protein substrate. When the transferred phosphate carries a radioactive isotope label, label incorporated into histone H1 reports catalytic action over the assay interval. It does not count CDK1 molecules, and a crude preparation can contain other protein kinases acting on histone H1. Attribution therefore needs controls.
A 2010 study prepared human HeLa S3 extracts after a double thymidine block, using swelling, a Dounce homogenizer, centrifugation, dialysis and concentration. Added recombinant cyclin B1 incubated for minutes.[ref]
Quantitative Reconstitution of Mitotic CDK1 Activation in Somatic Cell Extracts by Richard W. Deibler and others. Deibler and Kirschner (2010), Experimental Procedures, “Tissue Culture, Flow Cytomtery, and Somatic Cell Extract Preparation”; Figure 2A
Aliquots received histone H1 and terminal--labeled ATP for a -minute room-temperature reaction, stopped with Laemmli buffer. Dried SDS-PAGE gels exposed a phosphorimager screen; a BioRad Molecular Imager FX quantified labeling. No-added-cyclin B activity set the normalization baseline; RO-3306 reduced H1 activity approximately . Companion Western blots monitored Tyr15 phosphorylation, with actin Western blot loading control.[ref]
Quantitative Reconstitution of Mitotic CDK1 Activation in Somatic Cell Extracts by Richard W. Deibler and others. Deibler and Kirschner (2010), “H1 Kinase Assays”; Results, “A Three-Stage Activation Profile of CDK1 by Cyclin B”, reported Figure S2A inhibitor result; Figure 2B
Mixed extracts lose compartment boundaries; inhibitor sensitivity supports a major CDK1 contribution, with residual signal.[ref]
Quantitative Reconstitution of Mitotic CDK1 Activation in Somatic Cell Extracts by Richard W. Deibler and others. Results, “Establishing a Somatic Cell Extract System for Mitotic Entry”; “A Three-Stage Activation Profile of CDK1 by Cyclin B”
Substrate choice and inhibitor specificity limit attribution. The cell-cycle hysteresis experiment supplies a separate history comparison with this kind of histone H1 readout; its dose thresholds should not be transferred to human biological cells.
The histone H1 kinase assay: transferred label, controls and distinct protein measurements
. Original explanatory workflow of the inspected 2010 assay. The -minute extract incubation and -minute assay reaction are separate intervals. No gel lanes or measured intensities are simulated. The no-added-cyclin B baseline and RO-3306 perturbation have different purposes; neither a total-CDK1 Western blot nor a site-specific phosphorylation Western blot replaces the catalytic readout. Details.