MICRO-TAG® Protocols

1. Confirm you have: S-tagged target construct (or stable line), S-protein, RNA FRET substrate, assay buffers, and a real-time PCR instrument with fluorescence detection.

2. Decide your assay format: intact cells (captures permeability/context) or lysate (reduces permeability variables).

3. Choose plate format (96 or 384 well) and plan replicates (≥3 technical replicates per condition).

1. Pick a target from the validated catalog (recommended) or define a new target to evaluate.

2. Choose tag orientation (N- or C-terminal) based on expected domain accessibility.

3. Define experimental mode: Real-time temperature-series 

4. Define compound testing scheme:

• DMSO vehicle control
• Positive control ligand (if known)
• Dose series (e.g., 8–10 points, 3× dilution) 

5. Define temperature range and resolution (e.g., 8–16 steps across a suitable window).

(Skip if using a stable cell line.)

1. Seed cells into a 96/384-well plate to reach healthy density at assay time.

2. Transfect cells with the S-tagged target plasmid (and any required accessory plasmids).

3. Incubate 24–48 hours to allow expression.

4. Inspect cell health and confluence before proceeding.

Checkpoint: cells should look healthy with minimal detachment and consistent well-to-well density.

1. Prepare compound stocks at appropriate concentration in DMSO.

2. Create a dose series in assay-compatible buffer/media.

3. Prepare controls:

• DMSO-only
• No-target (untransfected or parental line)
• Optional: no S-protein or no substrate control (background)

4. Plan plate map with clear labeling for dose series and controls

1. Add compound doses (or DMSO) to cells.

2. Incubate long enough for intracellular equilibration (commonly ~30–90 minutes depending on cell type/compound).

3. Keep conditions consistent across the plate (timing, mixing, temperature).

1. Prepare detection mix fresh:

• S-protein at working concentration
• RNA FRET substrate at working concentration
• Assay buffer (optimized salt/pH)

2. Add detection mix to each well (same volume across conditions).

3. Mix gently to avoid bubbles (bubbles interfere with fluorescence).

4. Briefly equilibrate plate to starting temperature.
 

Checkpoint: background controls should remain low; target-expressing wells should show clear signal capability.

1. Load plate into a real-time PCR instrument configured for fluorescence readout.

2. Program a temperature series consisting of repeated cycles:

• Ramp to temperature step
• Hold for thermal equilibration
• Measure fluorescence (one or multiple reads)

3. Repeat across all temperature steps in the series.

4. Export raw fluorescence traces and temperature step metadata.
 

Notes:

- Use the same program across experiments to preserve comparability.

- Include DMSO controls at every temperature step.

1. For each well and temperature step, compute a signal metric:

• Slope of fluorescence vs time (common for real-time traces), or
• AUC over the measurement window.

2. Subtract background using no-target or no-reagent controls.
3. Normalize within temperature step if needed (e.g., to DMSO = 100%).
4. Aggregate replicate wells (mean ± SD).

1. At each temperature, plot metric vs compound concentration.

2. Fit a 4-parameter logistic curve to estimate EC50 per temperature (where applicable).
3. Flag temperatures where:

• Dynamic range is insufficient
• Curves are non-sigmoidal
• Background dominates

1. Combine temperature-step metrics to generate a global engagement profile:

• Summed/weighted metric across temperatures (or)
• A single composite readout representing total engagement effect.

2. Fit a global dose–response to estimate EC50(total).
3. Compare compounds by:

• EC50(total)
• Max effect (stabilization/destabilization amplitude)
• Shape of engagement profile across temperatures

1. Identify true cellular engagers (dose-dependent signal shift vs DMSO).

2. Compare compounds across a series to prioritize:

• Higher potency
• Stronger engagement amplitude
• Mechanistically distinct profiles

3. Decide next actions:

• Progress to functional assays
• Run lysate vs intact-cell comparison
• Expand temperatures or replicate

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