1. What is SDS-PAGE Molecular Weight Calculation?

The molecular weight calculation from SDS-PAGE uses a standard curve generated from proteins of known molecular weight to estimate the size of unknown proteins based on their migration distance (Rf value) in the gel.

2. How Does the Calculator Work?

The calculator uses the logarithmic relationship between molecular weight and migration distance:

Where:

• \( MW \) — Molecular weight (kDa)
• \( Rf \) — Relative front (migration distance of protein divided by migration distance of dye front)
• \( b \) — Y-intercept from standard curve
• \( m \) — Slope from standard curve

Explanation: The equation converts the linear relationship between log(MW) and Rf into the actual molecular weight.

3. Importance of Standard Curve

Details: Accurate molecular weight determination requires a properly constructed standard curve with at least 5 protein standards that span the expected molecular weight range of your samples.

4. Using the Calculator

Tips:

• Measure Rf values carefully (distance migrated by protein / distance migrated by dye front)
• Use the slope and intercept from your standard curve's linear regression
• Typical slope values range from -0.5 to -1.5 for most SDS-PAGE systems

5. Frequently Asked Questions (FAQ)

Q1: Why use log(MW) vs. Rf instead of MW vs. distance?
A: The logarithmic relationship provides better linearity across the wide range of molecular weights typically analyzed by SDS-PAGE.

Q2: What affects the accuracy of this calculation?
A: Gel composition, running conditions, protein modifications (glycosylation, phosphorylation), and protein shape can affect accuracy.

Q3: How many standards should I use?
A: Minimum 5 standards, ideally spanning the entire separation range of your gel.

Q4: What if my protein migrates abnormally?
A: Highly acidic/basic or membrane proteins may migrate anomalously. Consider alternative methods if suspected.

Q5: Can I use this for native PAGE?
A: No, this calculation is specific for SDS-PAGE where proteins are denatured and uniformly charged.