(a) Graph showing the comparative decrease in GFP fluorescence on GFP-TRAP-PC beads, and increase in the surrounding eluate, following exposure to UV light (UV filter block on TE2000U fluorescence microscope). (b) Images of GFP-TRAP-PC beads after incubation with GFP, pre- and post-cleavage by 30 s exposure to UV light, used to generate data for (a). (c) Western blot to quantify the binding and release capacity of GFP-TRAP-PC beads. 100 ng of GFP is completely immobilised on the beads. In this case,~60% of GFP is cleaved via UV exposure, corresponding to 3 ng x 20 = 60 ng GFP. (d) Western blots demonstrating the isolation and cleavage of Msd1-GFP and γ-Tubulin-GFP using GFP-TRAP-PC beads. The proteins, present in embryo extracts (input), are efficiently captured onto beads (bound), with ~50% released following UV exposure (post-cleaved beads and eluate). (e) Western blots of in vitro MT co-sedimentation assays using GFP-TRAP-PC beads. In the absence of taxol (-), Tubulin, pure Augmin (Msd1-GFP) and pure γ-TuRC (γ-Tubulin-GFP) remain in the supernatant (S). In the presence of taxol (+), Tubulin polymerises and is present in the pellet (P). Augmin and γ-TuRC co-sediment. GFP alone does not co-sediment with MTs in this assay. (f) Western blot of purified γ-Tubulin-GFP eluate, probed with anti-γ-Tubulin antibody. Endogenously expressed, untagged γ-Tubulin is co-purified with γ-Tubulin-GFP, presumably as part of active γ-TuRCs, at a ratio of ~3:1 γ-Tubulin-GFP: γ-Tubulin (as quantified by LiCOR analysis).