Native Gels for Detection of Large Complexes

1. Use use pre-cast 4-12% or 8-16% Tris-Glycine gradient gels, depending on the size of the complexes. The gels are from Invitrogen and are 1 mm thick with 10 or 12 wells. The running buffer is standard Tris-Glycine without SDS, pH 8.3.

2. If you are starting with glycerol gradient fractions, dilute them to ~5% final glycerol and load 10-15 ul per lane. Larger volumes seem to distort the bands. Alternatively, you can use native loading buffer from Invitrogen to prepare samples. Adding CHAPS to 2 mM and/or decreasing sample load may help if aggregation in the well is a problem.

3. Gels are run at 40V for 16 h at +4oC and electroblotted in Tris-Glycine buffer with 10% methanol for 6-8 h.

4. We use the Pharamacia HMW protein standards. These can be stained on the membrane after transfer with Sypro Ruby.