Step 5 · Experiment
Experiment designer
A structured starting point, not a protocol. Every suggestion is generated by the mock reasoning module from the selected gap and must be validated against your own SOPs.
Starting point
Lab capabilities
Feasibility notes adapt to what you can actually run.
Proposed experiment
Test: ERK1/2 → AS160
Hypothesis
The curated relationship ERK1/2 → AS160 is classified as limited in cardiomyocytes.
Suggested approach
- •Run a dose–response and time-course in the same cell model that produced your dataset.
- •Quantify total and phosphorylated protein by western blot with loading controls.
- •Measure transcript-level changes by RT-qPCR against two stable reference genes.
- •Profile oxygen consumption and extracellular acidification on the Seahorse XF analyser.
- •Run fluorometric uptake and viability assays in 96-well format.
- •Add a pharmacological inhibitor arm as a substitute for genetic loss-of-function.
Required controls
- •Vehicle-only control matched for solvent and volume.
- •Untreated baseline collected at each timepoint.
- •Inhibitor-only control without the primary treatment.
- •Positive control with a treatment of known effect size in this assay.
Readouts
- •Direct readout for ERK.
- •Direct readout for AS160.
- •Cell viability to rule out toxicity-driven artefacts.
- •A pathway-independent housekeeping readout to confirm assay specificity.
Expected outcomes
- •If the hypothesis holds: the downstream readout tracks the upstream perturbation in direction and magnitude.
- •If it fails: the downstream change persists when the upstream node is blocked, implying a parallel route.
- •Partial effects would suggest a contributing but non-exclusive mechanism — plan a combination arm.
Sample size guidance
Assuming the effect size seen in your dataset and typical assay variability, n = 6 independent biological replicates per arm gives ~80% power at α = 0.05 for a two-group comparison. Treat this as an planning estimate; run a formal power calculation with your own pilot variance before committing.
Feasibility
- •Runs with your declared capabilities: Western blot, qPCR, Seahorse XF, Plate reader, Cell culture.
- •No genetic perturbation declared: interpretation will rest on pharmacology, so include specificity controls.
- •Budget for at least two independent cultures to avoid batch-confounded replicates.
Generated by the mock reasoning module from curated demo data. Treat as a discussion starting point: verify reagents, timings, ethics approvals and statistics with your lab before running anything.