Figure 1. Preparation and harvest of the gradients. (a) Three different methods to prepare gradients as described in the text: the diffusion method (top left); the BioComp integrated rotational method (top right); and the pump with a gradient former method (middle). The different parts of the gradient are labeled: the dense solutions are shown dark, and solutions become lighter as the density decreases; (b) Manual gradient harvest. The top of the gradient is successively removed with an automatic pipette as described in the text; (c) A suction device constructed from a syringe or glass capillary, held in place with a ring‐like support (Plexiglas), the orifice of which is placed as close as possible to the bottom of the gradient. The syringe is connected with rubber tubing, first to a pump and then to a fraction collector. The fractions may also be collected manually.

Figure 2. Separation of spliceosomal complexes on a glycerol gradient. A splicing reaction was loaded onto a 10–30% (v/v) glycerol gradient in standard gradient buffer. The distribution of radioactively labeled pre‐mRNA was determined by Cherenkov counting.

Figure 3. The RNA and protein composition of a spliceosomal B complex were analyzed by electrophoresis, in an 8.3 M urea/9.6% polyacrylamide gel and by SDS‐PAGE, using a commercial pre‐cast gel (NuPAGE; Invitrogen), respectively. Proteins were visualized by Coomassie staining. The RNA composition of the spliceosomal B complex was visualized by silver‐staining [ 10]. While silver staining is the most sensitive stain for RNA, any other less‐sensitive stain can be used (e.g., methylene blue, toluidine blue O, or acridine orange). The radioactive RNA is detected by autoradiography on a PhosphorImager (GE Healthcare) or on X‐ray film (Kodak). Only the input pre‐mRNA is radioactively labeled and gives a signal upon autoradiography. Sizes of the RNAs: MINX‐Me pre‐mRNA: 324 nt; U2: 187 nt, U5: 116 nt (at least seven variants of this RNA are known; see Ref. [ 11] for details), U6: 106 nt. The doublet of the pre‐mRNA may be due to alternative structures of the pre‐mRNA, 3′ end trimming during the incubation, or to heterogeneity of the transcription template.