TAP-IPs

This protocol is a little different from other standard TAP IP protocols, but it is quite effective, especially when working with RNA binding proteins and looking for associated RNAs. Because many RNA binding proteins are found in stress granules, we adapted an approach described previously to generate cell extracts that recover such structures (Jain et al, 2016). Briefly:

1. For each TAP-tagged strain, harvest log-phase  cells from 1 L of culture (in YPD), or any other medium of interest. The cells can be stored at -80 C until ready.

2. Wash cells with distilled water.

3. Resuspend cells in 10 mL of lysis buffer (10mM Tris-HCl pH 7.5, 100mM sodium chloride, 1.5mM magnesium chloride, 0.5% NP-40), with 1:5,000 Antifoam emulsion and protease inhibitor cocktail added (and RNase inhibitor if looking for RNA-protein interactions), in Sarstedt 30 mL round bottom tubes with caps. 

4. Add 5 mL of glass beads and lyse the cells by 3 cycles of vortexing for 2 min followed by 2 min on ice.

5. Centrifuge the lysates at 850 g for 2 min at 4 C, and transfer the supernatants to standard 15 mL conical bottom tubes.

6. Add 0.2 mL of washed IgG Sepharose 6 Fast Flow beads (Millipore Sigma, Cat#: GE17-0969-01) to each sample and incubated on a rotisserie mixer for 0.5 h at room temperature.

7. Wash the beads 3 times with a buffer containing 10mM Tris-HCl pH 7.5, 100mM sodium chloride, 1.5mM magnesium chloride, 0.1% NP-40. Let the beads precipitate on ice, or briefly centrifuge at the lowest speed for 30 sec.

8. Resuspend the beads in 500 μL of the same buffer and stored at -80°, prior to RNA isolation and ddPCR.