What percentage should I use for a stacking gel? - (May/05/2012 )

Hello, all.

I've been trying to look through the forum for more information about what percentage to use when generating the stacking gel that goes on top of the running gel. As of late, I've been using a 5% stacking gel for just about any protein I've been examining as of late. I don't think this is appropriate. As such, I've been questioning if I should change up the percentage for the various protein molecules with varying molecular weight.

How should I know which stacking gel percentage to use?
When do I go about altering the percentage?

I use 5% for all of my stacking gels, and I would say that you probably don't want to change this too much. The idea with the stacking gel is that it helps you get better resolution with your protein bands. The proteins travel quickly through the stacker and then their run speed is drastically reduced when they hit the resolving gel, helping the sample to form a tight band before it enters into the resolving gel. This becomes especially important when your loading wells have larger volumes (>20 uL)

The part you really want to change is the resolving gel, and this really depends on the size of the protein you want to separate. For most proteins between ~25 kDa to ~75 kDa you probably want to use somewhere between 10% and 12%. If your protein is larger use a smaller percentage, and if it is smaller use a larger percentage. For complex samples that have many different sized proteins it is often helpful to use a gradient gel.