All you need to know when choosing a Gradient Gel

Why use a gradient gel?

If you’ve found yourself contemplating which type of polyacrylamide gel you wish to use for your western blotting protocol its worth having a read and learning about gradient gels.

Gradient gels are a great form of polyacrylamide gel to use during a western blot, especially for experiment which are focusing on the separations of proteins of similar molecular weight, e.g. Isomers.

So what is a gradient gel?

A gradient gel is a type of Stacking gel used for SDS-PAGE experiments but with a bit of a twist. Whilst regular Stacking gels are made with a lower concentration of polyacrylamide gel which is placed on top of a more concentrated gel layer, creating a sudden change in concentration intensity, a gradient gel on the other hand, is made so that there is a gradual dilution from a high to low concentration from the bottom region of the gel to the top.

If you are conducting an SDS-PAGE protocol which does in fact focus on proteins of a similar molecular weight you may encounter difficulties in visually differentiating closely spaced bands.

Gradient gels remove this complication as the gradual change in concentration throughout the polyacrylamide chamber has a direct effect on bands, which are only a couple of kD apart, migration ability during the PAGE process.

Gradient gels basics

The key feature of a gradient gel is that the higher concentration is casted at the bottom of the chamber by a Western Blot Gradient Maker. Then the concentration percentage begins to decrease to a lower known value towards the top. Creating a gradient in which your proteins have to work through.

When using a gradient gel it is essential you know or have a relative idea of your proteins molecular weight. This is in order to determine what the ideal gradient to make using the Gradient Maker for your western blot.

Should you make your own gel?

The process of creating your own gradient gel does, of course, extend the preparation time of your protocol in contrast to purchasing pre-cast Gradient gels. Pre-cast gels are the more popular option as they save time and resources. Nevertheless, they are more expensive and have a much shorter shelf life than you would expect.

Therefore it may in fact be worth putting in some extra time and tailor the casting towards your western blotting experiment.

If you are now looking to make a gradient gel yourself then please do continue onward.

Steps to casting your gradient gel

To make a gradient gel you will need to set up a Western Blot Gradient Maker, which is used to set the change in concentration as the gel cassette is filled. You can also do this by using two containers and tubing.

1. Set the first container (A) with the highest concentration of your not-yet-set polyacrylamide, and the second (B) with the lowest concentration of your gel. Note, each container should have an equal volume of polyacrylamide.
2. The containers then, need to be connected via a hose at the base. The two volumes should equate to the final volume you will need for the gel cassette.
3. Container A will need an additional hose to attach as its base, this should be fed into the cassette. Make sure to include a rapidly Rotating Mixer in the container to ensure an even dilution as the polyacrylamide from Container B enters. This is your self-made Gradient Maker.
4. Raise the two containers to that they are above the cassette, this allows gravitational-induced flow of the polyacrylamide from the containers to the cassette.
5. Switch the mixer on and open the hose for cassette A. Quickly open the hose between both containers A and B. Hydrodynamic pressure will keep the solution in both containers at the same level. This means the gel in container A will be diluted and replaced by B. Eventually the final outflow will entirely be the gel content from container B.
6. Finally, Once all the unset gel from both containers has completely passed into the cassette, leave the newly made Gradient gel to set and solidify.