Calculating the Rf values

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What is a Rf value?

Rf value is the distance that a component of interest has travelled, divided by the distance travelled by the solvent form. This value usually doesn't have a unit, because it is simply a ratio of two distances. It represents the rate at
which the components loaded onto the gel travel through it, relative to one another. 

The equation is as follows:   

Rf value calculated using dye front migration

Why do I need to calculate the Rf values?

The Rf value can be used to calculate the molecular weight of the protein of interest. To do this, running your sample on the same gel with molecular weight standards is necessary. 

This is because the conditions in which the gel runs affect the speed at which the proteins migrate.

Ensuring that the conditions are the same by loading the molecular weight standards onto the same gel means that the differences in the distance travelled are due to differences in molecular weight and, therefore, are representative of it. 

The fact that the exact conditions used determine the distance travelled also means that there are no set values for distances corresponding to molecular weight, so the standards have to be used along the sample every time.

After you run the gels, comparing the band of your protein to the known standard can give you a rough estimate, but calculating the Rf for both bands gives you a more precise number. 

The exact molecular weight can be found by plotting a graph of Rf value against molecular weight, adding to it the line of best fit, then using it to find the unknown molecular weight.