Protein Solubility Calculator — pH, pI & Salt Effect
Estimate how a protein's relative solubility changes with pH (relative to its isoelectric point), NaCl concentration, and temperature. Uses Cohn–Hofmeister principles for qualitative screening.
M
°C
Estimated relative to the same protein at pH = pI ± 2 and 0.15 M NaCl (normalised to 100)
- 1
|pH − pI|
|7.4 − 6.5| = 0.9 - 2
pH solubility factor
exp(0.25 × 0.9²) = 1.2245Gaussian model: solubility rises as pH moves away from pI. - 3
Salt (ionic strength) factor
10^(0.2 × 0.15) = 1.0715 - 4
Relative solubility vs optimal
(1.2245 × 1.0715) ÷ 2.9127 × 100 = 45
How does this calculator work?
Protein solubility is minimum at the isoelectric point (pI) where net charge = 0, and increases as |pH − pI| grows. NaCl below ~0.15 M gently boosts solubility (salting-in); above ~0.5 M it reduces solubility (salting-out; Cohn equation). This calculator estimates relative solubility from pH, pI, and NaCl concentration for qualitative screening — absolute values are protein-specific and require experiment.
Formula
How this is calculated
A protein's net charge — and therefore its electrostatic repulsion with neighbouring molecules — depends on the solution pH relative to its isoelectric point (pI). At pH = pI the protein carries zero net charge, inter-molecular electrostatic repulsion vanishes, and the tendency to aggregate or precipitate is highest, so solubility is at a minimum. Moving the pH away from the pI (either acidic or basic) increases net charge, increases repulsion, and increases solubility. This calculator uses a Gaussian model for the pH contribution and the Cohn–Edsall equation for the salt contribution.
Ionic strength from NaCl (a monovalent 1:1 electrolyte) equals the molar concentration: I = c_NaCl. At low ionic strength (below about 0.15 M) the added ions partially shield inter-chain charge repulsion, paradoxically improving solubility — this is the "salting-in" effect. Above roughly 0.15–0.5 M, the salt competes with the protein for hydration water, removing the solvation shell and reducing solubility — this is "salting-out", the basis of ammonium sulfate fractionation in protein purification. The Hofmeister series ranks ions by their relative salting-out potency.
Temperature effects are complex and protein-specific: most globular proteins are most soluble between 4 °C and 37 °C, but cold precipitation (cryoprecipitation) can occur below 4 °C, and heat denaturation above 50–60 °C can produce insoluble aggregates. The output is a qualitative relative index normalised to 100 at the estimated best conditions (pH = pI ± 2, I = 0.15 M). Absolute solubility (g/L) is highly protein-specific and must be determined experimentally.
Frequently asked questions
The pI can be predicted from amino acid sequence using free tools such as ExPASy ProtParam (web.expasy.org/protparam) — paste your sequence and read the theoretical pI. Experimentally, pI is determined by isoelectric focusing (IEF) gel electrophoresis or capillary isoelectric focusing (cIEF). Predicted pI values are typically within ± 0.5 pH units of the measured value for unmodified, folded proteins; post-translational modifications (phosphorylation, glycosylation) can shift pI significantly.
The Hofmeister series ranks ions by their ability to salt out proteins from solution. For anions: SO₄²⁻ > HPO₄²⁻ > CH₃COO⁻ > Cl⁻ > Br⁻ > SCN⁻ (left = stronger salting-out). Kosmotropic (water-ordering) ions on the left stabilise protein–protein interactions and promote precipitation; chaotropic ions on the right disrupt hydration shells and can even help dissolve aggregates. This calculator uses NaCl (Cl⁻), a mild kosmotrope, with K_s ≈ 0.30 M⁻¹. Ammonium sulfate, the gold standard for protein precipitation, has a much higher effective K_s (≈ 1.0–1.5 M⁻¹ per unit ionic strength).
Even at the isoelectric point, proteins retain some residual solubility because short-range hydrophobic and steric repulsions prevent complete aggregation under most conditions, and in practice the bulk pH distribution around a protein molecule is not perfectly uniform. The minimum solubility at pI can range from < 0.01 mg/mL for highly aggregation-prone proteins to several mg/mL for well-behaved globular proteins.
Also known as
TG we-Calculate Editorial Team. (2026). Protein Solubility Calculator — pH, pI & Salt Effect [Online calculator]. TG we-Calculate. https://we-calculate.com/calculator/protein-solubility-calculator
TG we-Calculate Editorial Team. "Protein Solubility Calculator — pH, pI & Salt Effect." TG we-Calculate. 2026. https://we-calculate.com/calculator/protein-solubility-calculator.
TG we-Calculate Editorial Team, "Protein Solubility Calculator — pH, pI & Salt Effect," TG we-Calculate, 2026. [Online]. Available: https://we-calculate.com/calculator/protein-solubility-calculator
@misc{wecalculate_protein_solubility_calculator, title = {Protein Solubility Calculator — pH, pI & Salt Effect}, author = {{TG we-Calculate Editorial Team}}, howpublished = {\url{https://we-calculate.com/calculator/protein-solubility-calculator}}, year = {2026}, note = {TG we-Calculate} }
Did this calculator help you?