Question 1

How is the extinction coefficient calculated?

Accepted Answer

The molar extinction coefficient at 280 nm is calculated using the Pace method: ε280 = (nTrp × 5500) + (nTyr × 1490) + (nCystine × 125), where nCystine = floor(nCys / 2). The Cys contribution assumes pairs of cysteines form disulfide bonds (cystines); if all cysteines are reduced, the cystine term is omitted. This method is accurate to within 5% for most proteins containing Trp residues.

Question 2

What is the difference between monoisotopic and average mass?

Accepted Answer

Monoisotopic mass uses the mass of the most abundant isotope of each element (e.g., 12C, 1H, 14N, 16O, 32S). Average mass uses the weighted average of all naturally occurring isotopes. Monoisotopic mass is used in high-resolution mass spectrometry, while average mass is appropriate for most biochemical calculations and corresponds to what you measure on a balance.

Question 3

How accurate is the pI prediction?

Accepted Answer

The Bjellqvist method predicts pI within ±0.5 pH units for most proteins. Accuracy depends on the protein's environment, post-translational modifications, and tertiary structure effects on pKa values. The calculation uses isolated amino acid pKa values and does not account for microenvironment effects. For precise pI determination, isoelectric focusing (IEF) experiments are recommended.

Question 4

How do I account for post-translational modifications?

Accepted Answer

Use the Modifications Panel to add common PTMs. N-terminal methionine removal subtracts 131.2 Da. Glycosylation adds approximately 2.5 kDa per N-glycan site (for complex-type glycans). PEGylation adds the specified PEG molecular weight. For other modifications, manually adjust the sequence or add the mass difference to the calculated MW.

Question 5

What if my protein has no Trp or Tyr residues?

Accepted Answer

If a protein lacks both Trp and Tyr residues, the extinction coefficient at 280 nm will be near zero (only Cys contributes via disulfide bonds). In this case, A280 measurements cannot reliably determine protein concentration. Alternative methods include BCA assay, Bradford assay, or measuring absorbance at 205 nm where peptide bonds absorb.

Question 6

How does disulfide bonding affect the extinction coefficient?

Accepted Answer

Each disulfide bond (cystine) contributes approximately 125 M⁻¹cm⁻¹ to the extinction coefficient at 280 nm. The calculator provides two values: one assuming all cysteines form disulfide bonds (nCys/2 cystines) and one assuming all cysteines are reduced (no cystine contribution). Under denaturing conditions with reducing agents (e.g., DTT, TCEP), use the reduced value.