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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 proteins on 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 works
The 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 the molecular 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 2 closely describes the relationship of the protein migration through the gel (R is close to 1 for each 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.
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Entering Rf values for HiMark™ standard proteins
1. 2. Click on the Calculator tab to open the data entry form. ™ 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 proteins
1. 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 curves for the individual MW ranges from the top and bottom portions of the gel. The HiMark Calculator was developed with Microsoft Office 2000 for Windows and it may ® not work properly with other versions of the Microsoft Office software. ©2003 Invitrogen Corporation. All Rights Reserved.

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0645 97 kDa 97.000 5.2041 116 kDa 116.6021 Rf Editable fields non editable Unknown Protein MW(Da) log10(MW) Rf © 2003 Invitrogen Corporation.3802 160 kDa 160.000 4.4624 240 kDa 240.000 4.000 5.000 4. .9868 66 kDa 66.6990 290 kDa 290.000 5. All Rights Reserved.HiMark Molecular Weight Estimation TM HiMarkTM Unstained HMW Standard Protein band MW(Da) log10(MW) 500 kDa 500.000 5.8195 55 kDa 55.7404 40 kDa 40.000 5.000 4.

800 1.200 Rf .4000 5.000 0.Whole Range 5.200 0.4000 4.0000 0.6000 5.600 0.2000 4.000 1.2000 Log MW 5.400 0.8000 5.0000 4.6000 4.8000 4.

0000 4.100 0.350 0.1000 5.080 0.060 0.160 0.220 Rf Low MW Range 5.500 0.200 0.1000 5.100 0.8000 5.250 0.400 0.9000 Log MW 4.000 0.5000 0.550 0.0000 0.6000 5.120 0.050 0.8000 4.High MW Range 5.600 Rf .000 0.2000 5.180 0.150 0.7000 4.040 0.200 0.450 0.300 0.3000 5.6000 4.4000 5.7000 5.020 0.140 0.5000 Log MW 5.