Protein pI & Molecular Weight

About Protein pI & Molecular Weight

This tool estimates a protein's isoelectric point, molecular weight and net charge at pH 7 from its amino acid sequence, and plots net charge from pH 0 to 14. The pI is the pH at which the molecule carries no net charge, which is useful for choosing buffers, ion-exchange conditions and isoelectric focusing ranges.

How to use it

1. Paste the one-letter protein sequence into the Protein sequence box. Non-letter characters are stripped.
2. Check the residue badge for the length that was read.
3. Read the Isoelectric point (pI), Molecular weight and Net charge at pH 7 tiles.
4. Use the Net charge vs pH bars to see where the protein crosses from positive to negative.

A 22-residue peptide

MASNTVHELLEKLGRDACEKWY

pI 5.37, molecular weight 2594 Da, net charge at pH 7 of -0.96

When to use it

• Choosing an anion or cation exchange column and buffer pH for purifying a recombinant protein.
• Predicting where a protein will focus on an isoelectric focusing strip or gel.
• Picking a buffer pH away from the pI to reduce precipitation risk.
• Comparing the charge of a wild-type protein and a point mutant.

Questions about Protein pI & Molecular Weight

How is the pI calculated?

Net charge is computed with Henderson-Hasselbalch for the N-terminus, C-terminus and the side chains of Lys, Arg, His, Asp, Glu, Cys and Tyr. The pH where that charge reaches zero is found by bisection between 0 and 14.

Which pKa values are used?

N-terminus 9.0, C-terminus 3.1, Lys 10.5, Arg 12.5, His 6.0, Asp 3.9, Glu 4.1, Cys 8.3 and Tyr 10.1. Other published tables give slightly different answers, so results will not match every calculator.

How is molecular weight calculated?

Average residue masses of the 20 standard amino acids are summed and one water added. Letters that are not standard residues, such as X, B or Z, contribute nothing.

Why might the measured pI differ?

Real proteins have modifications, cleaved signal peptides, disulfides and side chains whose pKa shifts inside the fold. Treat the estimate as a guide, often within about half a pH unit.

Related tools

• DNA to Protein TranslationTranslate a DNA or RNA sequence into protein across reading frames using the standard genetic code.
• ORF FinderFind open reading frames (ATG to stop) across all six frames, with position, length, and protein.
• Sequence StatsLength, base composition, GC%, and approximate molecular weight for raw or FASTA sequences.
• DNA ng / pmol / Copies ConverterConvert nucleic-acid mass, moles, and molecule count from length and type, with molarity.
• Codon OptimizerCodon-optimize a protein or coding sequence for E. coli, human, or yeast expression.
• Amino Acid Notation ConverterConvert amino acids between one-letter, three-letter, and full-name notation (auto-detected input).