DARTS Experimental Plan — DHEA
Drug Affinity Responsive Target Stability · Cellular Target Identification
Phases
Combining Prediction and Experimentation
Don't use a single method alone. Combining in silico prediction → narrowing the list → experimental confirmation (DARTS + CETSA) is the most effective strategy.
Key Materials
DARTS Principle
When DHEA binds to a target protein, the drug–protein interaction stabilizes the protein structure (thermodynamic stabilization). The target protein thus becomes more resistant to proteases compared to non-drug-bound proteins. After protease digestion, SDS-PAGE or MS is used to find bands/proteins that are more stable in the DHEA sample.
Stage 1 — Cell Lysate Preparation
Culture cells to appropriate density
Harvest 10⁷–10⁸ cells. Wash twice with cold PBS. Centrifuge 300 × g, 5 min, 4°C. Remove PBS completely.
Cell Lysis (Non-denaturing)
Add cold lysis buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40. Add PMSF 1 mM. Incubate on ice for 30 min, gentle agitation.
Important Note: Do not use protease inhibitor cocktail in the lysis buffer for DARTS experiments — only PMSF is needed. Full protease inhibitors will block the subsequent digestion step.