Translating Ribosomal Affinity Purification (TRAP)

Thomas et al. (2012) created stocks for expressing a GFP-tagged ribosomal protein under UAS control. Ribosomes from GAL4-expressing cells can be recovered by immunoprecipitation with an anti-GFP antibody and actively translated mRNAs isolated.

RiboTag Purification

Chen and Dickman (2017) and Huang et al. (2019) created similar stocks for expressing a FLAG-tagged ribosomal protein under UAS control and isolating ribosome-associated mRNAs using anti-FLAG antibodies.

Before using stock 79223, please note that a user has reported leaky expression of P{UAS-RpL3.FLAG}2 even when a GAL4 is not present. Results were confirmed by immunoprecipitation using anti-FLAG antibodies. No other tissues were examined in this analysis. With these results in mind, proceed with caution when using stock 79223 and consider using stock 77132 that carries the same P{UAS-RpL3.FLAG} construct inserted on the third chromosome. 

TU-tagging and EC-tagging

Miller et al. (2009) and Hida et al., 2017 generated stocks that express either uracil phosphoribosyltransferase (UPRT) or a cytosine deaminase (CD) – UPRT fusion under UAS control. In flies fed 4-thiouracil (4TU), 4TU is modified by UPRT and incorporated into newly synthesized RNA (TU-tagging). In flies fed 5-ethynylcytosine (5EC), 5EC is modified by CD:UPRT and incorporated into newly synthesized RNA (EC-tagging) . Thio- or ethynyl-labeled RNA can be selectively recovered by affinity purification.