I have seen many different protocols on making acrylamide gels for RDC measurements which include usually one of the following 1)drying the gel and re-swelling with protein solution 2)allowing equilibration of protein into an already swelled gel
It seems as though you can get a much higher protein conc. if you dry the gel so what is the advantage to letting it equilibrate?
asked Jan 24 '12 at 08:42
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.
answered Jan 30 '12 at 07:01
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).
answered Apr 19 '13 at 08:29
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:
answered Apr 24 '13 at 07:06