Understand the relationship
The reasoning behind the result
Relative migration normalizes a measured band position
Rf = dband/dfront
dband is migration measured from the top of the resolving gel to a band; dfront is migration from the same origin to the dye front. Their ratio Rf is dimensionless when both use the same unit and image scale. This workflow accepts bands between that origin and front, so Rf ranges from zero to one.
Uniformly enlarging an image multiplies both distances by the same factor and leaves Rf unchanged. A different origin, independently resized lane or mismatched dye-front measurement does not cancel correctly. Already calculated Rf records can be entered directly when their provenance is known.
SDS-PAGE calibration estimates an apparent mass
Matched SDS-PAGE standards provide a relationship between relative migration and known molecular masses under specified preparation and gel conditions. Over a suitable region, log₁₀ mass is approximately linear in Rf. The tool uses the actual standards entered for that run rather than assuming a universal curve.
An apparent mass inferred from migration can differ from true molecular mass because preparation, protein properties, modifications, incomplete denaturation and anomalous migration affect the comparison. It does not establish protein identity, composition or native complex size. Native PAGE requires a different interpretation and is outside this workflow.
Local interpolation and a global fitted range answer different assumptions
y = log₁₀(M); ylocal = y₁ + f(y₂ − y₁); f = (Rf − Rf₁)/(Rf₂ − Rf₁)
Local interpolation uses the two adjacent standards bracketing each band. The interpolated quantity is log mass, so the midpoint between 100 and 25 kDa estimates √(100 × 25) = 50 kDa, not the arithmetic midpoint of 62.5 kDa.
The fitted option uses all entered standards with equal weight in ordinary least squares: yfit = ȳ + b(Rf − R̄f). Residuals, RMS log residual and R² describe the entered fit. They do not establish a validated range, quantify measurement uncertainty or prove that a curved end region is appropriately linear. Choose the standards and range on the basis of the experiment.
Inverse predictions and range limits remain visible
Rf = Rf₁ + [(log₁₀ M − y₁)/(y₂ − y₁)](Rf₂ − Rf₁); dband = Rf dfront
For an entered target mass, inverse local interpolation or the inverse fitted line gives expected Rf within the measured calibration interval. When a dye-front distance is supplied, multiplying by that distance also gives expected migration in the selected image or length unit.
No extrapolation beyond the entered migration interval is produced. For a fitted line, inverse mass limits are the fitted endpoint values, which may differ from observed standard masses. An out-of-range sample remains in the result ledger with a status instead of a guessed number. Inverse migration does not predict a run time or confirm that a band will be detectable.
Follow the numbers
From measured distances to apparent mass and back
- A dye front lies 80 mm from the resolving-gel origin. The 100 kDa and 25 kDa standards lie at 16 and 48 mm, giving Rf values 0.2 and 0.6.
- An unknown at 32 mm has Rf = 32/80 = 0.4, halfway between those standards. Their log₁₀ masses are 2 and 1.397940009.
- The midpoint log mass is 1.698970004, so apparent mass is 10¹·⁶⁹⁸⁹⁷⁰⁰⁰⁴ = 50 kDa.
- For a 50 kDa target, the inverse calibration returns Rf 0.4 and a distance of 0.4 × 80 = 32 mm. Doubling all three distances in an enlarged image preserves the same Rf and apparent mass.
The calculation reconciles one matched relative-migration calibration; it does not determine the protein's identity or true molecular mass.
Quick guide
How to use this calculator
- Identify the SDS-PAGE run, standards and preparation conditions. Enter independently known masses from an appropriate matched standard set.
- Use distances from the top of the resolving gel to both bands and the dye front, or enter already calculated Rf values. Keep position origin and image scaling consistent.
- Choose local interpolation between adjacent standards or a declared fitted log-linear range. Enter named unknown migrations or target masses.
- Inspect the standards and every sample status. Treat the result as apparent mass under these conditions, separate from protein identification or an exact molecular mass.
Calculation method
Calculation and interpretation
Make an SDS-PAGE apparent-mass estimate traceable to its standards, dye-front normalization and chosen calibration range.
Rf = band migration / dye-front migration; y = log₁₀(M); y(Rf) = y₁ + [(Rf − Rf₁)/(Rf₂ − Rf₁)](y₂ − y₁); Mapparent = 10ʸ.
Worked example
From measured distances to apparent mass and back
The calculation reconciles one matched relative-migration calibration; it does not determine the protein's identity or true molecular mass.
Rf = band migration / dye-front migration; y = log₁₀(M); y(Rf) = y₁ + [(Rf − Rf₁)/(Rf₂ − Rf₁)](y₂ − y₁); Mapparent = 10ʸ.
Supported inputs
Precision and limits
SDS-PAGE apparent mass only
Standards, gel conditions and preparation must be matched independently. No protein identification, native-complex interpretation, anomalous-band diagnosis or exact-mass claim follows from the result.
Measurement and calibration boundaries
This calculator reads entered records, not an image. It does not select a ladder, infer the dye front or estimate uncertainty. Recorded Rf must be in 0–1; standards must have distinct positions and decreasing masses. Numerical inputs support 10⁻⁹ through 10⁹ in their selected units; explicitly nonnegative positions and times also accept zero. These are calculation limits, not operating recommendations.
Continue calculating
Related calculators
