TITLE: Co-immunoprecipitation in human cells
AUTHORS: Michael Tellier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol to perform co-immunoprecipitation (co-IP) in human cells. If the co-IP is followed by proteomics, it is better to remove the glycerol from the different buffers.</div></div>

[STEPS]
?. [Day 1]
Split human cells in a 150 mm dish to obtain a ~80% confluence on Day 2.Preblock Dynabeads protein A and/or G or Dynabeads M-280 Sheep anti-mouse IgG with 1 ml 0.5% BSA in PBS overnight on a rotating wheel at 16 rpm at 4°C. For Dynabeads protein A and G (30 mg/ml): use 40 μl of beads per condition (at least two: one IgG control and one IP). For Dynabeads M-280 Sheep anti-mouse IgG (10 mg/ml), use 120 μl of beads per condition.
?. [Day 2]
Beads preparationWash the beads three times on ice with a magnetic rack with 600 μl of IP buffer (25 mM Tri–HCl pH 8.0, 150 mM NaCl, 0.5% NP-40, 10% Glycerol, 2.5 mM MgCl2, and protease inhibitor cocktail). Perform the washes by gently mixing by inversion.Add 2-5 μg of antibody to the beads in 600 μl IP Buffer.Incubate for at least 2 hours on a rotating wheel at 16 rpm at 4°C.
?. [Day 2]
LysisCells should be ~80% confluent.Prepare 8 ml of ice-cold PBS with protease inhibitor cocktail per condition.Wash cells twice with ice-cold PBS.Scrap the cells in 8 ml of ice-cold PBS prepared in step 2 of 2.1 and transfer to an ice-cold 15 ml Falcon tube.Centrifuge at 500 g for 5 minutes at 4°C.Remove the supernatant and resuspend the pellet in 800 μl Lysis Buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 10% glycerol, 2.5 mM MgCl2, protease inhibitor cocktail, phosphatase inhibitor, 1× PMSF, and 25–29 units of Benzonase) by pipetting up and down ~ 10 times and transfer in an ice-cold 1.5 ml tube.Incubate on a rotating wheel at 16 rpm for 30minutes at 4°C.Centrifuge at 13,000 g for 15 minutes at 4°C.Transfer the supernatant in a new ice-cold 1.5 ml tube. Add 800 μl of Dilution Buffer (150 mM NaCl, 10% glycerol, 2.5 mM MgCl2, protease inhibitor cocktail, phosphatase inhibitor, and 1× PMSF) and mix by pipetting up and down ~ 8-10 times.Perform Bradford or another protein quantification method to determine the concentration of each sample.Freeze at -20°C at least 100 μl of sample to be used as Input.
?. [Day 2]
IPWash the beads three times on ice with a magnetic rack with 600 μl of IP buffer.Add 1 mg of proteins to the 1.5 ml tubes containing the washed Ab-conjugated beads (top to 600 μl with IP buffer).Incubate on rotating wheel at 16 rpm for 2 hours in a cold room.Wash the beads three times on ice with a magnetic rack with 600 μl of IP buffer (first wash: 5 minutes incubation on a rotating wheel at 16 rpm; no incubation for the second and third washes).Wash the beads three times on ice with a magnetic rack with 600 μl of IP buffer without NP-40 (first wash: 5 minutes incubation on a rotating wheel at 16 rpm; no incubation for the second and third washes).Do a quick spin down (3 seconds at 800 rpm) and put back the tubes on the magnetic racks to remove the last drops.The beads can be freeze at -20°C to perform protein digestion on beads for proteomics or to perform later the elution.
?. [Day 2]
Elution with LDS for western blots1. Add 36 μl 1x LDS + 4 μl DTT (1M stock solution) to each tube, and resuspend the beads by vortexing with a low strength.2. Incubate on a Thermomixer for 10 minutes at 70°C at 1,400 prm.3. Perform a quick spin and put the tube on a magnetic rack.4. Transfer the supernatant to a fresh protein Lobind 1.5 ml tube. Use 20 μl per western blot (one elution = two western blots).5. Store at -20°C. Add DTT again before loading on a gel (2 μl  per 20 μl  sample).
?. [Day 2]
Elution with glycine for proteomicsAdd 1.5x volume of 0.2M glycine solution at pH 2.3 to the beads. Resuspend the beads by pipetting up and down.Incubate on a Thermomixer for 7 minutes at 25°C at 1,000 rpm.Perform a quick spin and put the tube on a magnetic rack.Transfer the supernatant to a fresh protein Lobind 1.5 ml tube.Neutralise by adding 1/10th of the volume of 1M Tris-HCl pH 8.5.Repeat the glycine elution once (steps 1 to 5, part 2.4).Pool both fractions in the same tube.