Using R_f value to calculate molecular weight

In a previous post, we looked at how to calculate the R_f value of a protein from a western blot. The formula is:

R_f = Migration distance of the protein

Migration distance of the dye front

So why do we need the Rf value?

You can use the R_f value to calculate the molecular weight of a protein of interest. If you want to do this, you will need to run your sample on the same gel as a set of molecular weight standards (proteins of known molecular weight.)

1. Run your western blot with your protein of interest and the molecular weight standards. You can find some helpful tips on western blotting here.

2. You can estimate the molecular weight of your protein of interest by simply comparing the position of its’ band on the gel with those of the protein standards. However, to get a more precise idea of the molecular weight of your target protein, you will need to calculate the R_f values of both the protein standards and your target protein, using the formula above.

3. Next, you will need to plot a standard curve. Standard curves are created by plotting known data – in this case, the Rf values of the molecular weight standards. The standard curve can then be used to estimate the value of an unknown quantity: the molecular weight of the target protein.

Draw the standard curve by plotting the R_f values of the protein standards on the X axis against the molecular weight on the Y axis.

4. Draw a best-fit line through the points. You should have a linear plot – if not, you may need to go back and check your western blot method.

5. Use the standard curve to determine the molecular weight of your protein of interest.

There are just a few more things to remember:

• The protein standards and your protein of interest must undergo electrophoresis on the same gel, under identical conditions.
• When a protein gives a thick band on the gel, try to measure from the middle of the band when calculating R_f.
• The stronger the linear relationship shown by your standard curve, the more accurate your determination of molecular weight can be.

While there are a few other ways to determine the molecular weight of a protein, using R_f values from a western blot is by far the most accessible; the method relies on equipment and techniques that are well-known and commonly available.

Are you looking for antibodies for your western blot? St John’s laboratory has over 30,000 primary and secondary antibodies for you to choose from. Browse our catalogue here.