Estimate protein molecular weight using migration distance (Rf) and log-linear regression, mimicking GraphPad Prism SDS-PAGE analysis.
Reference Standards (Calibration)
The known molecular weight of your larger standard protein.
Please enter a valid positive number.
Distance traveled by Marker 1 from the well.
Please enter a valid positive number.
The known molecular weight of your smaller standard protein.
Please enter a valid positive number.
Distance traveled by Marker 2 from the well (must be further than Marker 1).
Please enter a valid positive number.
Unknown Sample
Distance traveled by your protein of interest.
Please enter a valid positive number.
Estimated Molecular Weight
0.00
kDa (Kilodaltons)
Parameter
Value
Slope (Log MW / mm)
–
Y-Intercept (Log MW)
–
Log(MW) of Target
–
Formula Used: Log(MW) = Slope × Distance + Intercept
● Standard Curve (Log-Linear)
● Target Unknown
What is the Prism Molecular Weight Calculator?
The Prism Molecular Weight Calculator is a specialized tool designed for biochemists and molecular biologists to estimate the molecular mass of proteins based on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) data. While software like GraphPad Prism is traditionally used for this complex regression analysis, this calculator provides a quick, web-based alternative for immediate estimation without needing heavy desktop software.
This tool utilizes the principle that the migration distance (or Relative Mobility, Rf) of a protein in a gel is inversely proportional to the logarithm of its molecular weight. By using two or more standard markers to establish a calibration curve (log-linear regression), we can accurately interpolate the size of an unknown protein band.
Researchers using Western Blots or Coomassie-stained gels often use this logic to confirm if their target protein has been expressed or isolated correctly. It eliminates the guesswork of visually comparing bands.
Prism Molecular Weight Formula and Explanation
The core mathematical principle behind the Prism molecular weight calculator is the linear relationship between the migration distance and the base-10 logarithm of the molecular weight.
The formula for the regression line is:
y = mx + b
Where:
y = Log10(Molecular Weight)
x = Migration Distance (mm) or Rf value
m = Slope of the line (usually negative, as larger proteins migrate less)
b = Y-intercept (theoretical Log MW at 0 distance)
Variable Definitions
Variable
Meaning
Unit
Typical Range
MW
Molecular Weight
kDa
10 – 250 kDa
x (Distance)
Migration from well
mm
0 – 100 mm
Slope (m)
Rate of separation
Log(kDa)/mm
-0.01 to -0.05
Practical Examples (Real-World Use Cases)
Example 1: Identifying a Purified Enzyme
A researcher runs a gel to identify an enzyme expected to be around 45 kDa. They use a standard ladder.
Marker A: 75 kDa travels 15mm.
Marker B: 25 kDa travels 45mm.
Unknown Band: Travels 30mm.
Result: The calculator determines the slope is negative. Since 30mm is exactly halfway between 15mm and 45mm, the Log(MW) will be halfway between Log(75) and Log(25). The estimated MW comes out to approximately 43.3 kDa. This confirms the band is likely the target enzyme.
Example 2: QC of Antibody Fragments
A lab is testing for antibody heavy chains (approx 50 kDa).
Marker High: 100 kDa at 10mm.
Marker Low: 20 kDa at 60mm.
Unknown Band: Travels 25mm.
Calculation: The Prism molecular weight calculator establishes the standard curve. The input of 25mm yields a result of roughly 63 kDa. This suggests the protein might be glycosylated or is not the heavy chain, prompting further investigation.
How to Use This Prism Molecular Weight Calculator
Measure Your Standards: accurate measurement of the migration distance (in mm) from the top of the resolving gel to the center of the band for at least two known markers.
Enter Marker Data: Input the Molecular Weight (in kDa) and Distance (mm) for a larger marker (Marker 1) and a smaller marker (Marker 2).
Measure Your Unknown: Measure the distance of your target protein band.
Input Unknown Distance: Enter this value into the "Target Band" field.
Analyze Results: The calculator will display the estimated MW in kDa and plot the point on the generated regression chart.
Key Factors That Affect Molecular Weight Results
Several physical and chemical factors can influence the accuracy of your Prism molecular weight calculator results:
Gel Percentage: Higher percentage acrylamide gels resolve small proteins better, while low percentage gels are for large proteins. Using the wrong percentage changes the linearity of the standard curve.
Voltage & Heat: Running a gel too fast generates heat, which can cause "smiling" effects where bands migrate differently in the center vs. edges, distorting distance measurements.
Glycosylation: Post-translational modifications add mass but also affect charge and shape. SDS-PAGE assumes charge-to-mass ratio is constant, but glycoproteins often migrate slower (appearing larger) than their true polypeptide weight.
Buffer Composition: The pH and ionic strength of the running buffer affect the stacking and resolving of bands.
Protein Shape: This calculator assumes proteins are fully denatured by SDS and reducing agents (like DTT). If a protein retains secondary structure, it will not migrate solely based on MW.
Measurement Error: Using a ruler vs. digital software to measure distance in millimeters introduces human error. Digital quantification is recommended for inputs.
Frequently Asked Questions (FAQ)
Can I use this for DNA ladders?
No. DNA migration is also logarithmic, but the charge-to-mass ratio is different, and the units are Base Pairs (bp), not Daltons. You should use a specific DNA calculator.
Why does the result differ from the manufacturer's label?
Manufacturer labels are approximations. Differences in gel chemistry (Tris-Glycine vs Bis-Tris) can cause apparent shift in MW. Always rely on your internal standard curve.
What if my unknown distance is outside the markers?
This is called extrapolation. It is less accurate than interpolation. For best results, choose markers that bracket your unknown protein (one larger, one smaller).
Does this replace GraphPad Prism?
For simple linear regression of two points, yes. However, GraphPad Prism offers non-linear regression and can handle many more standard points for higher precision.
What unit should I use for distance?
You can use mm, cm, or pixels, as long as you are consistent across all inputs. The ratio (slope) depends on the unit, but the final MW calculation cancels the unit out.
Why is the chart logarithmic?
Protein migration in a polymer mesh is not linear; it follows a log decay. Plotting Log(MW) makes the line straight, allowing for simple linear algebra to solve for the unknown.
Is the intercept value important?
The Y-intercept represents the theoretical Log(MW) of a protein that doesn't migrate at all (Distance = 0). It is a constant used for the calculation logic.
Can I use more than two markers?
This simplified web tool uses a two-point calibration. For multi-point calibration, calculate the average slope or use statistical software.
Related Tools and Internal Resources
Molarity Calculator – Calculate solute mass for specific molar concentrations.