Current Protocols in Chemical Biology 2011 (Volume 3) by Adam P. Arkin

By Adam P. Arkin

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The HsPUM1-HD (hereafter named PUM1) was designed to match the RNA sequence of ND6, which was connected with EGFP fragments (Fig. 1B). Upon interacting with RNA, PUM1 brings the fragments of EGFP into an orientation and proximity in which they are sufficiently close for the fragments to associate and fluoresce. Monitoring the fluorescence signals allows spatial and temporal analysis of mRNA localization in individual living cells. , 2009). PUM1, fused to either the N-terminal or the C-terminal halves of split mCitrine, was engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV).

Basic Protocol 1 describes a smallmolecule treatment procedure, and Basic Protocol 2 an siRNA transfection procedure. Following one of these protocols, the assay continues with fixation, permeabilization, staining, and imaging, as described in Basic Protocol 3. Assay validation data generated using this protocol for both siRNA and small molecule controls and Z determination is shown in Figures 1 and 2, respectively. This assay procedure can be adapted to the measurement of other targets using different primary antibodies and other modifications, such as changes in fixation, permeabilization, and blocking conditions.

When the cells are 90% confluent, transfect the cells with plasmids GN-mPUM1, VN-mPUM1, mPUM2-GC, or mPUM2-VC using Lipofectamine 2000 according to the manufacturer’s instructions. 3. Incubate the cells 48 hr at 37◦ C. 4. Lyse the cells by adding 1 ml of ice-cold lysis buffer A. 5-ml tube. 5. Centrifuge the cells 3 min at 15,000 × g, 4◦ C, and collect the supernatant. 6. Add anti-flag antibody (1/1000 v/v) or anti-GFP antibody (1/2000 v/v) into the solution and incubate for 1 hr at 4◦ C with a rotator.

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