How to calculate the Rf value of a protein from western blot

How to calculate the Rf value of a protein from western blot

Western blot is a common tool for identifying, quantifying and determining the size of specific proteins. Essentially, western blotting allows a mixture of proteins to be separated using gel electrophoresis. The proteins are then transferred from the gel to a membrane (the “blotting” part). The protein of interest is marked by washing the membrane with a solution containing a primary antibody. Then a secondary antibody that can be visualised (through staining or immunofluorescence) is added. The final result is – hopefully – a clear image with defined bands showing the target protein.

Calculating the Rf value

The Rf (retardation factor) value is the ratio of the distance moved by the solute to the distance moved by the solvent. The term originates in chromatography, where it was observed that a particular compound will always travel the same distance in a particular solvent, as long as conditions are constant.

In western blot, calculating the Rf value is useful because it allows the molecular weight of a protein to be determined. The formula is:

Rf = Migration distance of the protein
Migration distance of the dye front

To work out the Rf value by hand (rather than using software), follow these simple steps:

1. Measure the distance your protein of interest has travelled, in millimetres. Make sure you know which way round your image should be, so that you measure the correct distance! For example, if the anode (+) was at the bottom of the gel, the negatively- charged proteins will have moved from the top towards the bottom of the gel.

2. Measure the migration distance of the dye front i.e. how far the dye has moved from the top of the gel. Once you have measured this once, you can use it to calculate Rf values of different proteins in your sample.

3. Use the formula above to calculate the Rf value. As the top value will always be larger than the bottom value, your result will be less than 1.

And that’s it! As you can see, calculating Rf is relatively straightforward. However, you may want to use the Rf values from your western blot to determine the molecular weight of your protein of interest. We will look at how to do this in the next post.