| Protein | pI | pH | Salt | Temp (°C) | Conc (mg/mL) | Hydrophobicity | Expected trend |
|---|---|---|---|---|---|---|---|
| Lysozyme | 11.0 | 6.0 | NaCl 150 mM | 25 | 10 | 0.35 | Higher solubility away from pI |
| IgG-like antibody | 8.4 | 6.0 | NaCl 50 mM | 4 | 50 | 0.55 | Aggregation risk increases with concentration |
| Enzyme (typical) | 6.2 | 7.5 | KCl 200 mM | 25 | 5 | 0.45 | Moderate salt screens interactions |
| Membrane protein | 6.8 | 8.0 | NaCl 150 mM | 20 | 2 | 0.75 | Needs detergent to stay soluble |
- Δ = pH − pI estimates net-charge direction and distance from pI.
- A Gaussian NearPi = exp(−( |Δ| / 0.85 )²) captures how close you are to pI.
- Predicted solubility scales with screening (moderate salt) and drops with salting-out (very high salt).
- Hydrophobicity and concentration add penalties; stabilizers add boosts.
- Ceiling ≈ 120 × Π(factors), then clamped to 0.5–250 mg/mL.
- Solubility ≈ Ceiling × (1 − 0.35 × NearPi^sharp).
- Risk increases near pI, with hydrophobicity, temperature, and concentration.
- Enter your protein’s pI, molecular weight, and an estimated hydrophobicity.
- Set the buffer, pH, salt type, salt level, and temperature.
- Add stabilizers (glycerol/arginine/detergent) if you expect aggregation.
- Click Predict solubility to view results above the form.
- Use CSV/PDF export to document screening conditions and outcomes.
What the predictor estimates in practice
This calculator estimates an expected soluble concentration (mg/mL) under your chosen buffer conditions, plus an upper “ceiling” and two quality indicators: aggregation risk and a native‑friendliness stability index. The output is designed for screening, where you compare options rather than accept a single value as final.
For planning, treat numbers as order‑of‑magnitude guides. A predicted solubility of 30 mg/mL suggests routine handling, while values under 5 mg/mL often need optimization. Use small-volume trials: 6–12 pH points, 3 salt levels, and two additive mixes. Record clarity, centrifugation losses, and recovery after filtration. When the confidence score is lower, expand screening and confirm with light scattering or SEC. Repeat at 4°C and room temperature to capture true storage behavior, not only immediate solubility outcomes.
Why pH relative to pI dominates results
Proteins often show their lowest solubility near the isoelectric point. The model uses a near‑pI index that increases as |pH−pI| approaches zero. If your Δ(pH−pI) is within 0.5, the tool will usually predict lower solubility and higher aggregation risk, especially at higher concentration.
Salt and ionic strength: screening ranges that matter
Moderate ionic strength can reduce unfavorable electrostatic interactions and improve handling. The calculator treats 0–500 mM added salt as a useful screening window and applies a stronger salting‑out penalty beyond roughly 400 mM. Salt type is handled as a practical modifier for early planning.
Temperature and concentration: compounding effects
Raising temperature can increase unfolding and collision frequency, which can raise aggregation probability. Concentration adds crowding and self‑association. The predictor therefore reduces effective solubility as you increase mg/mL, and it can flag cases where your entered concentration approaches the predicted ceiling.
Additives that commonly shift outcomes
Glycerol (often 5–15%) supports conformational stability. Arginine (50–200 mM) is widely used to suppress aggregation in development workflows. Low detergent levels can help hydrophobic or membrane‑adjacent targets remain dispersed. Urea may improve apparent solubility but lowers the stability index.
How to interpret the Plotly pH sweep
The Plotly curve recalculates predicted solubility across a pH range centered on your pI (typically pI±2). Use it to pick pH candidates with higher predicted solubility, then validate with a solubility assay and basic stress tests. Export the chosen conditions to document decisions.
Is this prediction accurate for every protein?
No. It is a heuristic screening estimate. It helps compare conditions and identify risk drivers, but it cannot replace solubility measurements for your specific construct and purification history.
What should I do if pH is close to pI?
Move pH at least 0.5–1.0 units away from pI and recheck. If activity requires that pH, test stabilizers, moderate salt, and lower concentration to reduce aggregation.
How do I choose a hydrophobicity value?
Use an estimated 0–1 score from your sequence analysis or prior experience. Higher values indicate more hydrophobic surfaces and typically lower solubility, especially without additives.
Why can high salt reduce predicted solubility?
Very high ionic strength can promote salting‑out, reducing water available to solvate the protein. The calculator applies a stronger penalty when salt is far above common screening ranges.
Does urea always improve solubility?
It can raise apparent solubility by disrupting interactions, but it may compromise native structure and activity. The tool reflects this tradeoff by boosting solubility while lowering stability.
Which output should guide my next experiment?
Use predicted solubility to rank conditions, aggregation risk to prioritize safeguards, and the stability index to avoid harsh formulations. Then validate the top few conditions experimentally.