Protein Molecular Weight, Isoelectric Point and Extinction Coefficient
6 min read · Updated June 10, 2026
A protein sequence quietly encodes a handful of physical properties you need every week in the lab: how heavy the protein is, what pH it carries no net charge at, and how strongly it absorbs UV light. This guide explains where molecular weight, isoelectric point and the extinction coefficient come from and how to put them to work.
Molecular weight
A protein's molecular weight is the sum of its amino-acid residue masses plus one water molecule (the residues are joined by losing water at each peptide bond). Using average isotopic masses gives the figure quoted by tools like ExPASy ProtParam, usually reported in daltons (Da) or kilodaltons (kDa).
Molecular weight tells you where a band should run on an SDS-PAGE gel, how to convert between mass and moles of protein, and roughly how big the molecule is. Bear in mind that post-translational modifications, bound cofactors and disulfide bonds can shift the real mass away from the sequence-based estimate.
Theoretical isoelectric point (pI)
The isoelectric point is the pH at which the protein carries no net charge. Below its pI a protein is net positive; above it, net negative. It is calculated by adding up the charge contributions of the ionisable groups — the N- and C-termini and the side chains of Asp, Glu, Cys, Tyr, His, Lys and Arg — at each pH using their pKa values, then finding the pH where the total is zero.
The pI guides ion-exchange chromatography (which resin and buffer pH to use), predicts where a protein may aggregate (solubility is often lowest near the pI), and helps interpret 2D gels and isoelectric focusing.
Extinction coefficient and protein concentration
Proteins absorb UV light at 280 nm mainly because of tryptophan and tyrosine (and, to a small degree, cystines). The molar extinction coefficient ε₂₈₀ can be estimated directly from the counts of these residues using the Edelhoch/Pace method: ε = 5500 × nTrp + 1490 × nTyr + 125 × n(cystine).
With ε you can turn an A280 reading into a concentration via Beer's law (A = ε × c × path length). The related A(0.1%) value — the absorbance of a 1 g/L solution in a 1 cm cuvette — lets you read concentration in mg/mL straight off the spectrophotometer: concentration (mg/mL) = A280 / A(0.1%).
GRAVY and other quick descriptors
The GRAVY score (grand average of hydropathy) averages Kyte–Doolittle hydropathy values across the sequence; a positive value suggests a more hydrophobic protein and a negative value a more hydrophilic one. Together with amino-acid composition and the counts of positively and negatively charged residues, these descriptors give a fast first impression of a protein before any wet-lab work.
Frequently asked questions
- Why does my protein run at a different size on a gel than its calculated weight?
- SDS-PAGE migration depends on more than mass — highly charged, glycosylated or unusually shaped proteins can run faster or slower. The sequence-based molecular weight is the mass of the unmodified polypeptide.
- How do I get concentration from A280?
- Divide your A280 reading by the A(0.1%) value to get mg/mL (for a 1 cm path length), or use the molar extinction coefficient with Beer's law to get molar concentration. Choose the reduced or cystine extinction value depending on whether disulfides are formed.