Determining Molecular Weight from a Standard Curve of Mobility of Protein Bands
The most accurate way to do this is to determine Rf, defined as, Rf (retention front), the relative mobility of protein bands in a gel.
Relative mobility as Rf is determined by the following ratio: Rf = distance migrated by protein/distance migrated by dye front
The marker for relative mobility can be the dye front or a low-molecular-weight standard protein. With gradient gels the dye front is often diffuse or runs off the bottom of the gel, so selecting an internal protein marker is helpful. The use of an internal marker (dye or protein) corrects for lane-to-lane differences in mobility.
Alternatively, the simplest approach to estimating molecular weights is to use migration distance into the gel without converting to a relative mobility. This simplified approach can be used for this investigation.
- Determine the migration distance into the gel of the standard and unknown proteins using a millimeter ruler. Record the distance migrated by the standards in the ladder as centimeters or millimeters in an Excel spreadsheet.
- Determine the log10 of each of the molecular weights of the standards.
- Using the Chart Wizard in Excel, plot migration distance in centimeters or millimeters (or use relative mobility, Rf, if you want) on the y-axis. Plot log10 of the molecular weight of the standards on the y-axis. Remember that Excel makes the data column on the far left the x-axis. You can use Excel’s default option, the linear scale, since you have already converted to log values for molecular weight as your data points.
- Add a trendline to perform linear regression of the data. Use the options menu to display the r2 value and equation.
- The purpose of plotting the data and performing the regression is to generate a linear curve through the standards so that the size of the unknown can be estimated. Thus, choose a region of the plotted data that is reasonably linear for performing the regression. Gradient gels usually show wide-range linearity so you will probably be able to use all the data points to generate the line.
- If your regression line has an r2 value of .95 or greater, use the equation of the line to estimate the size of the unknown proteins of interest.
The general equation of a straight line is: y = mx + b
where m is the slope and b is the y-intercept. In this case, the equation becomes: mobility = (slope)( log molecular weight) + y-intercept
Determining the size of an unknown becomes straightforward simply by placing the unknown or sample protein mobility (y) into the equation and calculating x (log molecular weight) and making a conversion to molecular weight.
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