HiMark™ Molecular Weight Calculator

for Invitrogen NuPAGE® Tris-Acetate Gels

Protein molecular weight on SDS-PAGE is usually estimated by plotting Rf values versus log molecular weight and is calculated using a linear equation. We found that migration of proteinson a variety of SDS-PAGE gels is more accurately calculated using non-linear equations. The HiMark™ Molecular Weight Calculator allows you to easily and accurately calculate the molecular weight of your proteins on NuPAGE® Novex 3-8% or 7% Tris-Acetate Gels using the standard curve obtained with HiMark™.

To use the calculator, click on the Calculator tab.

How the program worksThe linear range for molecular weight estimation is confined to the middle part of the gel. The separation of the large proteins on the top and small proteins on the bottom of the gel does not follow a linear dependence. Attempts to plot a straight line through all points on the entire molecular weight range of the gel will lead to errors in the calculation of molecular weight for high and low molecular weight proteins. We found that polynomial curve fits the data with higher accuracy than a straight line. It is possible to find one equation that calculates the molecular weight of low and high proteins over the entire calibration curve, however the trendline fit is much better if the molecular weight ranges are plotted separately.

To increase the accuracy of molecular weight estimation and find an equation that best fits themolecular weight range, we separated each gel into two parts based on the molecular weight of proteins – high molecular weight (top half of the gel), medium and low molecular weights (bottom half of the gel). The two parts of the gel are described by a separate equation that closely describes the relationship of the protein migration through the gel (Reach equation) and is used to calculate accurate molecular weight for unknown proteins using the best fit of the high molecular weight standard curve.

To view the charts click on the Charts tab.

Note: This spreadsheet is designed for use with NuPAGE® Novex Tris-Acetate Gels. Different gel types exhibit different molecular weight separation and the equations used are accurate only for the particular gel type used to create them. To avoid estimating erroneous molecular weights, do not use a spreadsheet designed with data from one gel type for molecular weight estimation on another gel type.

Entering Rf values for HiMark™ standard proteins1. Click on the Calculator tab to open the data entry form.

2. Enter the Rf values for the HiMark™ standard by typing the values or pasting the data from an image analysis program in the Rf column (light green highlighted area).

The charts (see Charts tab) will be updated with the new values.

Entering Rf values for unknown proteins1. Enter the Rf values for the unknown proteins in the Rf column for the unknown proteins.

2. Press ENTER or click on another cell after entering the last number.

3. Click "Calculate" button.

The molecular weight for unknown proteins is calculated using equations from the best-fit

Protein band MW(Da) log10(MW) Rf500 kDa 500,000 5.6990 Editable fields290 kDa 290,000 5.4624 non editable240 kDa 240,000 5.3802160 kDa 160,000 5.2041116 kDa 116,000 5.064597 kDa 97,000 4.986866 kDa 66,000 4.819555 kDa 55,000 4.740440 kDa 40,000 4.6021

UnknownProtein MW(Da) Rf

© 2003 Invitrogen Corporation. All Rights Reserved.

HiMarkTM Molecular Weight Estimation

HiMarkTM Unstained HMW Standard

log10(MW)

0 2 4 6 8 10 12

4.0000

5.0000

6.0000

7.0000

8.0000

9.0000

10.0000

11.0000

f(x) = 0R² = 0.000000

Whole Range

Rf

Lo

g M

W

0.3 2.3 4.3 6.3 8.3 10.3 12.30.0000

2.0000

4.0000

6.0000

8.0000

10.0000

12.0000

f(x) = 0R² = 0.000000 Low MW Range

Rf

Lo

g M

W

0.11 2.11 4.11 6.11 8.11 10.11 12.110.0000

2.0000

4.0000

6.0000

8.0000

10.0000

12.0000

f(x) = 0R² = 0 High MW Range

Rf

Lo

g M

W