I have heard that people use the soaking into a swelled gel method to avoid introducing inhomogeneity in the gel due to drying. I use the drying method, but I have seen that it can cause the gel to get a bubble in it.

Hi Andrew,

It matters what kind of gel you are trying to prepare, i.e. either a compressed gel (where you set the plunger/piston in the shigemi tube to a certain level and then let the gel swell with the protein solution) or a stretched gel (where you use an apparatus to get the gel swollen with protein solution into the NMR tube). So, if it is compressed, I think one has to use a dry gel, while if it is stretched one has an option to do it two ways as you have mentioned.

I do agree with the previous answer that swelling a dried gel with protein solution might result in inhomogenous alignment (which could be inferred from the line shape of resonance lines).

Waj

I've tried both, and find I prefer the drying method, and I never had any homogeneity problems with it. You do have to be careful to get the dried gel out of the casting chamber intact, and the gels are sticky so we always dried them on a teflon plate.

Three other practical notes:

1. I always soaked the cast gel in several changes of water before drying to remove any unpolymerized acrylamide and polymerizing agents.
2. The gel has to be completely dry before soaking. A 37-50 degree oven/incubator helps with that.
3. It's a good idea to pre-soak the dry gel for just a few minutes with the same buffer your protein sample is in before adding your real protein sample, then pour it off and add the protein. This seems to help prevent your protein getting supersaturated and potentially crashing out as the water rushes into the gel. After that, I soak overnight before stretching into an NMR tube.