Western Blot / SDS-PAGE Calculator
Calculate protein molecular weight from SDS-PAGE band position using standard curve.
Why Log-Linear Interpolation
Proteins migrate through the polyacrylamide mesh at a rate inversely related to the logarithm of their mass, not their mass directly. Plotting log(MW) against migration distance therefore gives a straight line, and unknowns are read from it.
Two consequences follow. Interpolation between bracketing markers is reliable; extrapolation beyond the marker range is not, because the linear region ends where proteins either fail to enter the gel or run with the dye front.
Resolution is also uneven. A given gel percentage resolves well over roughly a tenfold mass range and poorly outside it, which is why the acrylamide percentage should match the target protein.
When Apparent Mass Is Wrong
Discrepancies between predicted and observed mass are common and usually informative rather than errors.
| Cause | Direction | Typical magnitude |
|---|---|---|
| N-glycosylation | Runs larger | 2–3 kDa per site, sometimes far more |
| Phosphorylation | Slightly larger | 1–2 kDa per site — charge effect |
| Highly acidic proteins | Larger | Bind less SDS per residue |
| Proline-rich, highly basic | Smaller | Anomalous SDS binding |
| Incomplete reduction | Smaller | Compact disulfide-bonded form |
| Proteolysis | Smaller | Discrete lower band |
Glycosylation is the commonest explanation for a band running well above prediction. PNGase F treatment collapses the band toward the predicted mass, which both confirms the cause and gives the core protein size.
Multiple Bands
| Pattern | Likely explanation |
|---|---|
| Ladder of evenly spaced bands | Glycoforms with differing chain numbers |
| Band at exactly twice the mass | Incomplete denaturation — SDS-resistant dimer |
| Discrete lower band | Proteolytic fragment or splice variant |
| Smear | Degradation or heavy heterogeneous glycosylation |
A band at precisely double the expected mass usually means the sample was not fully denatured. Some proteins form SDS-resistant oligomers, and boiling can occasionally make this worse for membrane proteins by promoting aggregation — incubating at 37°C rather than 95°C is standard for those.
Worked Examples
Common Mistakes
The log-linear relationship holds only between the smallest and largest markers. Bands outside that range cannot be sized reliably.
Glycosylation, phosphorylation and unusual amino acid composition all shift apparent mass. A discrepancy of 10–20% is common and often expected.
Heating to 95°C promotes aggregation of many multipass membrane proteins. Incubating at 37°C for 30 minutes usually gives cleaner results.
That signature indicates an SDS-resistant dimer from incomplete denaturation rather than a distinct protein.