Separation of Drosophila RNA Silencing Complexes by Native Gel Electrophoresis

Large, multicomponent complexes mediate many stages of eukaryotic gene expression, from transcription to translation. Despite their size, these complexes and their precursors can often be resolved and analyzed by native gel electrophoresis. Although other techniques exist for large-scale separations, native gels require very little sample, making them superior for analytical applications. We have developed a native gel system for rapidly and reliably separating complexes that form on short interfering RNAs (siRNAs) (1 ). This approach has facilitated our analysis of RNA silencing complexes and holds promise for additional mechanistic studies, particularly those that are limited by sample size [such as experiments with mutant lysates (2 )]. In this chapter, we describe the procedures involved in reproducibly detecting Drosophila siRNA/protein complexes and discuss significant issues as well as techniques for optimization.