Figure 2.

Resolving different polymerases in “native” 2D gels (run-ons in [³²P]UTP included).

(a) Resolving complexes II + III with Coomassie blue in the first dimension. The cartoon shows regions selected for mass spectrometry analysis. First, an autoradiograph of the gel was prepared; overlapping spots of (nascent) [³²P]RNA are along the diagonal. ~0.03% protein, ~0.8% DNA, and ~5% nascent [³²P]RNA initially present were contained in the region indicated (dotted outline). After blotting, the membrane was stained with Ponceau S; most protein is on the diagonal. Next, the membrane was immuno-probed successively for three polymerases (using antibodies against RPA194, RPB1, and RPC62); the three are partially resolved. Note that complex I is destabilized by the Coomassie blue in the first dimension, and so migrates rapidly.

(b) Resolving complex I (no Coomassie in either dimension). The cartoon shows regions selected for mass spectrometry analysis. First, an autoradiograph was prepared; overlapping spots of (nascent) [³²P]RNA are again along the diagonal. After staining with Coomassie, spots are seen to overlap regions rich in [³²P]RNA. After blotting, the membrane was probed for the polymerases (as above); complex I now runs the slowest.

(c) Proteins in regions indicated in a and b were resolved on a 4-15% SDS-acrylamide gel, and stained with Coomassie.