Fig. 1

Construction of an Arabidopsis E2 protein library with an N-terminus biotin tag using a wheat germ cell-free system. a Flow chart of the wheat germ-based procedure for the high-throughput production of an Arabidopsis E2 library with an N-terminus biotin tag. The first step involves the high-throughput preparation of DNA templates for transcription using 2-step “split-primer” PCR, followed by in vitro transcription using phage-coded SP6 RNA polymerase, and finally translation using the wheat germ cell-free system. All the steps were carried out in 96-well microtiter plates. b Immunoblot analysis of N-bio-E2s expressed by the wheat germ cell-free system. For analysis, 2–6 μL crude recombinant E2 proteins with N-terminus biotin tag were loaded onto SDS-PAGE and detected by streptavidin-HRP antibody. A total of 35 out of 37 predicted Arabidopsis E2s were detected. Arrows on the figure show the expected signal for each E2 and asterisks refer to the E2s used later in vitro ubiquitination analysis (Fig. 4, Fig. 5, Fig. 6)