Nuclear extraction and co-immunoprecipitation protocol

Nuclear extraction

  1. Pellet cells (1-10M), wash 1x with ice cold PBS
    • Use fresh pellet, or freeze and store at -80°C
  2. Resuspend pellet in cold buffer, incubate on ice for 10min
    • 100-1000µl depending on pellet size, 500µl for 10M cells
Cytoplasmic Buffer (4°C)Stock Concentration50ml (49.25ml H2O)
10mM NaCl5M100µl
10mM Tris1M500µl
3mM MgCl21M150µl
Protease inhibitor tablet 1/2
  1. Centrifuge at 500rcf for 5min at 4°C
  2. Pipette remove supernatant (cytoplasmic fraction)
  3. Resuspend pellet in Mod. RIPA + Benzonase (Bzn) + MgCl2
    • 100µl for 10M cells
    • Prepare 5ml Mod. RIPA with ½ tablet of protease inhibitor
    • E.g., for 500µl Mod. RIPA with protease inhibitor, add 2µl Bzn and 0.5µl 1M MgCl2
Modified RIPA-0.1% (4°C)Stock Solutions50ml (40.5ml H2O)
0.1% NP-40/IGEPAL10% (RT)0.5 ml
0.1% Sodium deoxycholate (LIGHTSENSITIVE!)1% (RT, protected from light)5 ml
150mM NaCL5M (RT)1.5 ml
50mM Tris pH7.5 (pH8)1M (RT)2.5 ml
Protease inhibitor tablet add right before use
1U Bnz/µl Buffer250U/µl (-20C; 4µl per 1ml Buffer)add right before use
1mM MgCl2 (1X)1M (1000X, RT)add right before use
  1. Incubate on ice for 20-30min
  2. Centrifuge at 14000rpm for 15min at 4°C to remove cell debris
  3. Transfer supernatant to a pre-chilled tube (nuclear fraction)

Co-immunoprecipitation

Day 1:
  1. Save 5% input
  2. Thaw anti-FLAG M2 beads on ice and gently invert to thoroughly resuspend resin
  3. For IP from 10M cells, pipette slowly to transfer 50µl anti-FLAG M2 beads to eppi
  4. Place on magnetic stand to remove supernatant (50% glycerol)
  5. Wash 4x with 500µl PBS + 0.01% Tween-20 + protease inhibitor by placing eppi on and off magnetic stand (do not need to pipette resuspend)
    • Prepare 10ml PBS + 10µl 10% Tween-20 + 1 protease inhibitor tablet
  6. Resuspend beads in 400µl Mod. RIPA + protease inhibitor and add 100µl lysate (500µl total)
  7. Rotate overnight in cold room
Day 2:
  1. Centrifuge briefly and place eppi on magnetic stand to remove supernatant
    • Save supernatant in eppi as needed
  2. Wash 2x with 500µl MCLB for 10min each, rotating in 4°C
MCLBStock Concentration5ml (4.4ml H2O)
150mM NaCl5M150µl
50mM Tris pH7.5 (pH8)1M250µl
0.5% NP-4010%250µl
Protease inhibitor tablet 1/2
  1. Add 50µl 100ng/ml 3xFLAG-peptide to beads
    • Dilute 1µl 5mg/ml stock in 49µl 1xTBS (without protease inhibitor)
  2. Incubate at RT for 1h on orbital shaker
  3. Centrifuge briefly and place eppi on magnetic stand
  4. Transfer supernatant to a pre-chilled eppi
  5. Add 4xLDS + 1mM DTT (100mM stock) and incubate at 70°C for 10min
  6. Load input and IP on gel