Protein Properties Calculator

Calculate protein properties: charge, instability index, aliphatic index, and secondary structure tendency.

Number of positively charged residues (R+K+H)

Number of negatively charged residues (D+E)

Protein length (total amino acids) (aa)

Number of Ala (A) residues

Number of Val+Ile+Leu (V+I+L) residues

Please check your inputs and try again.

What Isoelectric Point Predicts

The pI is the pH at which a protein carries no net charge. Below it the protein is positively charged; above it, negative. This single number determines behaviour in almost every purification and separation technique.

pH relative to pI	Net charge	Consequence
pH < pI	Positive	Binds cation exchange resin
pH = pI	Zero	Minimum solubility — may precipitate
pH > pI	Negative	Binds anion exchange resin

Solubility is lowest at the pI because there is no net charge to drive electrostatic repulsion between molecules. This is exploited deliberately in isoelectric precipitation, and it is also why buffers are chosen at least one pH unit away from the pI during purification.

Extreme pI values reflect function. Histones have pI near 11 because they are rich in arginine and lysine — the positive charge is what binds them to the phosphate backbone of DNA. Pepsin has a pI around 1, allowing it to function in gastric acid.

Aliphatic Index and Thermostability

AI = X_Ala + 2.9X_Val + 3.9(X_Ile + X_Leu)

The aliphatic index measures the relative volume occupied by aliphatic side chains. Higher values correlate with thermostability, because bulky hydrophobic residues pack the protein core tightly and resist unfolding.

Aliphatic index	Interpretation
< 70	Lower thermostability
70–90	Typical mesophilic protein
> 90	Elevated — common in thermophiles

Proteins from thermophilic organisms consistently show higher aliphatic indices than mesophilic homologues, which is one of several compositional signatures of thermal adaptation alongside increased ion pairing and reduced surface loops.

Note these are all sequence-derived predictions. Actual pI can shift by a unit or more from the calculated value, because buried residues have altered pK_a values and post-translational modifications such as phosphorylation add charge the sequence does not reveal.

Worked Examples

Example 1: Histone H4: highly positive charge

Rich in Arg+Lys, few acidic

Result: pI ~11 — binds negatively charged DNA

Histones: ~20% Lys+Arg

Example 2: Acidic protein: net charge -20

Many Asp+Glu, few basic

Result: pI ~4-5 — low pI

Calreticulin, calsequestrin: Ca2+ binding

Example 3: Choosing the ion exchange mode

Protein with pI 5.5, buffer at pH 8.0

Result: Anion exchange

Above the pI the protein is negatively charged, so it binds a positively charged anion exchange resin. Below pI 5.5 the choice would reverse.

Example 4: Why histones are so basic

pI near 11, rich in Arg and Lys

Result: Binds the DNA phosphate backbone

At physiological pH a histone carries strong positive charge, which is exactly what allows it to neutralise and compact negatively charged DNA.

Common Mistakes

⚠️

Purifying at a pH close to the pI

Solubility is minimal there and the protein may precipitate. Work at least one pH unit away, and choose the ion exchange mode accordingly.

⚠️

Treating calculated pI as exact

Buried residues have shifted pK_a values and modifications such as phosphorylation add charge. Measured pI often differs by a unit or more.

⚠️

Assuming a high aliphatic index guarantees stability

It correlates with thermostability but does not determine it. Disulfide bonds, ion pairs and overall fold contribute at least as much.

⚠️

Ignoring post-translational modifications

Phosphorylation adds negative charge and glycosylation alters surface properties. Sequence-based predictions cannot account for either.

Frequently Asked Questions

pI calculation?

pH where protein net charge=0. Above pI: negative (excess of bases). Below pI: positive. Important for: protein purification (ion exchange chromatography at specific pH), gel electrophoresis, solubility (lowest at pI), precipitation.

Aliphatic index?

(Ala×2.9+Val×3.9+Ile×4.5+Leu×4.5)/length×100. Higher value: more nonpolar, thermostable. Thermostable proteins from thermophiles have high aliphatic index. Used to predict thermostability without structure.

What does the isoelectric point tell you?

The pH at which the protein has no net charge. It determines which ion exchange resin to use and the pH at which solubility is lowest.

Why is solubility lowest at the pI?

Because there is no net charge to generate electrostatic repulsion between molecules, so they aggregate more readily. This is the basis of isoelectric precipitation.

What is the aliphatic index?

A measure of the relative volume of aliphatic side chains. Values above 90 are common in thermophilic proteins and correlate with thermostability.

How accurate is calculated pI?

Approximate. Buried residues have shifted pK_a values and post-translational modifications alter charge, so measured values often differ by a pH unit or more.