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ProtocolsYSD Pipeline
FACS Staining, Sorting & Enrichment
Standard operating procedure from the WeaveSeq experiments team, shared across the YSD pipeline runs.
Standard operating procedure from the WeaveSeq experiments team, shared across the YSD pipeline runs.
Stain the induced yeast library for simultaneous display detection and target binding, then sort by FACS to enrich for high-affinity, specific binders across multiple rounds of increasing stringency.
| Channel | Fluorophore | Detects | Laser |
|---|---|---|---|
| Channel 1 (Expression) | anti-c-Myc-FITC (or AF488) | Full-length display via C-terminal c-Myc tag | 488 nm |
| Channel 2 (Binding) | Streptavidin-PE or SA-AF647 | Biotinylated target binding | 488/561 or 633 nm |
This two-color approach normalizes binding signal to expression level, enabling discrimination of true affinity differences from expression-level artifacts.
Harvest: Centrifuge 2,000–3,000 × g, 5 min, 4°C. Count cells.
Wash: Wash 3× with 1 mL ice-cold PBSA. Resuspend pellet completely each time. This removes residual media proteins that compete for non-specific binding.
Pre-complex target + SA (recommended): Mix biotinylated target + SA-fluorophore at 3:1 molar ratio in PBSA. Incubate ≥1 h at RT, protected from light. Use 200 nM target + 67 nM SA-AF647 as starting point.
Primary incubation: Resuspend cell pellet in 50–100 µL PBSA containing:
Incubate 4°C (on ice), 1 h, with gentle agitation (rotator or flick every 15 min). For Kd <1 nM, extend to 16–20 h overnight. ⚠️ Keep all steps at 4°C after primary incubation to minimize dissociation of weak binders.
Wash 3× with 1 mL ice-cold PBSA. Quick, gentle spins (3,000 × g, 2 min).
Secondary incubation (if using unconjugated anti-c-Myc): Resuspend in 50–100 µL PBSA containing:
Resuspend in 500 µL–1 mL ice-cold PBSA.
Filter through 40 µm cell strainer immediately before sorting to remove clumps.
Keep on ice, protected from light, until analysis (sort within 2–4 h).
| # | Control | Staining | Purpose |
|---|---|---|---|
| 1 | Unstained | No reagents | PMT voltage setup; autofluorescence baseline |
| 2 | Secondary only | SA-fluorophore + anti-mouse-FITC (no target, no primary Ab) | Non-specific secondary binding |
| 3 | Expression only | Anti-c-Myc-FITC only (no target) | Display induction check; set expression gate |
| 4 | No-target binding | Anti-c-Myc + SA-fluorophore (no biotinylated target) | SA binding to yeast surface |
| 5 | Negative clone | Full stain on non-binding clone or empty vector | Binding baseline |
Gate the no-target control such that ≤0.1–0.3% of cells fall in the binding-positive gate. This defines the false-positive rate.
Apply gates in this order during sorting:
All events
→ FSC-A vs SSC-A: Gate yeast population (exclude debris)
→ FSC-H vs FSC-W (or SSC-H vs SSC-W): Exclude doublets
→ Live/Dead gate (optional, PI⁻ or SYTOX Blue⁻)
→ Expression gate: c-Myc⁺ (FITC/AF488⁺)
→ Binding gate: Target⁺ (PE/APC/AF647⁺)
Within the c-Myc⁺ population, plot Binding (y-axis) vs Expression (x-axis). Cells on the main diagonal have binding proportional to expression. Cells above the diagonal have higher binding per displayed molecule = higher affinity.
Binding ^ / Sort Gate
(AF647) | / (top 0.5–5%)
| /
| / (diagonal)
| /
| /
| /
| /
| /
| /
|/________________>
Expression (FITC)
Gate shape: Diagonal or trapezoid collecting the upper region of the double-positive population. Use a square/orthogonal gate for maximum dynamic range (Kowalsky et al. 2015, PLOS ONE).
| Round | [Target] | Gate % | Goal |
|---|---|---|---|
| 1–2 | 100–500 nM (saturating) | 5–15% | Eliminate non-binders; preserve diversity |
| 3–4 | 10–50 nM (~EC₅₀) | 1–5% | Affinity pressure begins |
| 5–6 | 1–10 nM (<Kd target) | 0.1–2% | Fine discrimination |
Lever 1: Decreasing target concentration. Halve or quarter [target] each round after round 2. Do not drop >10× in a single step—you risk eliminating all binders.
Lever 2: Tighter gates. Progressively narrow from 10% → 5% → 2% → 0.5%.
Lever 3: Off-rate selection (rounds 4+).
Lever 4: Alternating fluorophores. Swap SA-PE ↔ SA-AF647 between rounds to avoid enriching fluorophore-binding clones.
Always sort ≥10× more cells than the estimated remaining diversity.
| Round | Estimated Unique Clones Remaining | Min. Cells to Sort | Min. Cells to Stain |
|---|---|---|---|
| 1 | 10,000 (input library) | 100,000 | 1 × 10⁶ |
| 2 | 2,000 | 20,000 | 2 × 10⁵ |
| 3 | 500 | 5,000 | 5 × 10⁴ |
| 4+ | 50–200 | 1,000–5,000 | 1–5 × 10⁴ |
Sort cells directly into SD-CAA (not SG-CAA — glucose represses GAL1 so cells stop expressing displayed protein and focus on growth).
Add 1× Pen-Strep to suppress bacterial contamination from sheath fluid.
Grow at 30°C, 250 rpm until saturation (typically 2–3 days for ≥10⁵ cells; smaller populations need longer outgrowth).
Re-induce (SOP 05) before the next sorting round.
After each sort, freeze a backup aliquot (SD-CAA + 15% glycerol, −80°C).
| Parameter | Setting |
|---|---|
| Nozzle size | 100 µm (yeast are ~5 µm; 70 µm possible but higher clogging risk) |
| Sheath pressure | Low (20–25 psi for 100 µm nozzle) |
| Sort mode | Purity (for enrichment sorts) or Single Cell (for monoclonal) |
| Event rate | ≤7,000 events/sec (higher rates reduce purity) |
| Temperature | 4°C (cooled sample chamber) |
Follow Ackerman et al. (2009) Biotechnol Prog 25:774–783:
This removes ~99.8% of reagent-binding clones in ~4 hours and achieves up to 30,000-fold enrichment in a single pass.
For specificity against related proteins, use dual-color FACS:
| Check | Method | Criterion |
|---|---|---|
| Display efficiency | c-Myc⁺ % | ≥30% (consistent across rounds) |
| Enrichment | % binding⁺ vs previous round | Increasing |
| Population shift | Overlay histograms | Binding population emerging |
| Diversity retention | Analytical FACS (pre-sort) | No single clone dominating |
| Viability | FSC/SSC or PI⁻ | ≥80% viable |