1. Grow cells to ~80% confluency. You will need ~2 million cells/RIP, or ~4 million cells for IgG (neg control) & Ago2 RIP
2. Rinse cells with HBSS (volume = culture medium)
3. If cells attached, release in trypsin-EDTA ~5 min and quench with 4-5 volumes serum-containing medium
4. Count cells, aliquot 2 million cells/ tube, and pellet at 4 oC, 1000rpm, 5 min. Discard supernatant.
5. Wash 2x with ice cold PBS (volume = medium removed).
6. Discard supernatant & add 200µL mild or harsh lysis buffer + Protease Inhibitor Cocktail (PIC) (P8340) + Ribonuclease Inhibitor (RNase inhibitor)(R1158)/cell pellet
7. Incubate on ice 15' (or store o/n at -80 oC)
8. Pellet debris at 4 oC, 16,000g, 10 min
9. Transfer sup to fresh tube
10. Remove 10 µL from each cell lysate for 5% input and store on ice
1. Add 40µL Protein A Magnetic Beads to 200µL wash buffer in 1.5mL tube for 2 RIPs (IgG & Ago2)
2. Place tubes on magnetic stand & remove liquid
3. Wash 1x 200µL wash buffer
4. Resuspend in 200µL wash buffer
5. Add 5 µg rabbit Anti-Rat IgG (whole molecule) antibody produced in rabbit (R9255)
6. Incubate at RT with rotation for 30 min.
7. Spin briefly & remove liquid on magnetic stand
8. Wash 2x with 1ml wash buffer.
9. Resuspend in 200µl wash buffer & split into 2 x 100µL
10. Add 2.5 µg rat IgG (I4131) or Monoclonal Anti-AGO2 antibody produced in rat, clone 11A9 (SAB4200085).
11. Incubate at RT with rotation for 30 min.
12. Spin briefly & remove liquid on magnetic stand.
13. Wash 2 times with 0.5mL wash buffer.
14. Remove wash buffer completely on magnetic stand.
1. Prep IP Buffer:
1. Resuspend bead pellets in 0.2mL IP Buffer.
2. Verify 5% input reserved from each cell lysate and stored on ice
3. Add 0.1 mL IP Buffer/cells in mild lysis or 0.6 mL/cells in harsh lysis
4. Transfer diluted lysate to resuspended beads
5. Incubate at 4 oC with rotation O/N
1a. For mild washing, wash 5x by adding 1mL wash buffer, vortex, spin briefly, collect beads on magnet, & remove
1b. For harsh or stringent washing, wash 1x 1ml wash buffer, 2x 1mL stringent wash buffer, and 2x 1mL standard wash buffer
2. After last wash, resuspend in 0.2mL wash buffer
3. Bring reserved input samples to 0.2mL with wash buffer
1. Add 0.5mL Tri Reagent (T9424) to each 0.2mL RIP or input
2. Add 0.1mL chloroform (C2432) to each, vortex thoroughly, and spin at 4 oC, 16,000g, 10 min.
3. Prepare 1.5mL tubes containing:
6 µL linear acrylamide (Ambion; 5mg/mL)
60 µL 5M ammonium acetate (09691)
600 µL 2-propanol (I9516)
4. Transfer aqueous to tubes from above, vortex, & precipitate at -80 oC at least 1 hour
5. Thaw tubes from -80 oC on ice
6. Spin at 4 oC, 16,000g, 10 min.
7. Carefully pour off supernatant
8. Wash once with 0.5ml 70% ethanol
9. Spin at 4 oC, 16,000g, 10 min.
10. Carefully pour off sup and dry pellets in a laminar flow hood
11. Rehydrate each in 10µl RNase-free water