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Control of translation initiation plays a critical role in the regulation of gene expression in all organisms, yet the mechanics of translation initiation in eukaryotic organisms are not well understood. Confounding studies of translation are the large number and overlapping functions of many initiation factors in cells, and a lack of cap-dependence in many in vitro systems. To shed light on intricate mechanisms that are often obscured in vivo, we use a fully reconstituted translation initiation system for analyzing RNA interactions with eukaryotic translation initiation factors and complexes from the model organism Saccharomyces cerevisiae. This system exhibits strong cap dependence, and dependence on translation factors varies with mRNA 5′ UTR sequences as expected from genome-wide studies of translation. Here we provide optimized protocols for purification and analysis of the effects of labeled and unlabeled …