Co-immunoprecipitation from S2 cells

S2 cell IP Protocol using magnetic beads

50 mM Hepes                       5.95 g Hepes

150 mM NaCl                        4.39 g NaCl

1 mM EDTA                          1 ml of 0.5 M EDTA

0.5 mM EGTA                       0.5 ml of 0.5 M EGTA

0.9 M glycerol                       41.5 g glycerol (weigh this first)

                                                500 ml pH 7.4 

IP from S2 cells

1. Prepare buffer solutions. For IP of 8 samples I make up 10ml of buffer with inhibitors (for 1x concentration of inhibitors) and split that in half to have 5ml of buffer + inhibitors and makeup 5ml of lysis buffer.

Lysis buffer:

            10ml  IP Buffer

            1 tablet Complete Mini stock (from 4 degree)

            1 tablet of PhosSTOP (from 4 degree) 

            5ul µL 1M DTT (add at the end)

            50 µL10% Tx100

            50 µL 10% Sodium Deoxycholate monohydrate (made fresh, .1g/1mL dH20) 

2. Resuspend cells within the well and collect 2 ml of cells, spin them 3 times at 4° 1500rpm for 30sec rotating the tubes 180° after each spin to pack the cell pellet, then aspirate the supernatant. Wash 1X in 250 µL Hepes Buffer alone – no inhibitors or detergents then spin same as before at 4° 1500rpm 3 times for 30sec, then aspirate supernatant again.

3. Lyse cells in 250 µL of lysis buffer by passing 10X through a tuberculin syringe (27 gauge needle). I count pulling solution in AND out of syringe as 1 passage. Put on rocker (nutator) in the Cold Room for 15 min to further lyse cells.

4. Spin down cells in the 4°C microfuge at 14K for 10min. Save 10 µL of the supernatant (pre-IP lysate) to compare protein levels between your samples or efficiency of IP. (you could also solubilize the left over pellet if you believe not all the protein is being released by the lysis).

5. During spin down, prepare antibody tubes. For pull down using GFP antibody, I use 1:1250 GP-⍺-GFP. I aliquot 0.8 µL of 1:10 GP-⍺-GFP working stock into fresh, pre-chilled tubes. Label tubes for lysates, respectively. 

6. Transfer supernatant to tubes with antibody and let incubate while rocking in 4ºC cold room for 30min.

7. While lysate + antibody are incubating, prepare beads.

• Mix the stock tube to resuspend the magnetic beads, don’t vortex just swirl.

• Add 50μl of 1X cell lysis buffer to the magnetic bead pellet. 
• Transfer 10 μl of bead slurry to each clean tube. 

8. Spin lysate + antibody down at 5k for 20sec 4º

9. When lysate + antibody are ready and spun down, place tubes with magnetic beads in magnetic separation rack for 10-15seconds, then carefully pipet out the lysis buffer and transfer lysate + antibody to beads and mix one at a time (don’t want beads to dry out). Rock at 4º for 1hr 

10. Short spin for 5sec to collect all of solution. Take out 10ul for post-IP sample if necessary

11. Wash beads 1x in 250µL of lysis buffer, place in magnetic separation rack to pipet out buffer. Then wash 1x in 250µL IP Buffer + inhibitors, place in magnetic separation rack to pipet out buffer.

12. Add 10ul of 2X SDS loading buffer to 10ul pre-IP lysates and post-IP samples

13. (except for samples you plan to treat with phosphatase) Add 40 µL of 1X SDS loading buffer to beads (make up 1x SDS by diluting 2x SDS stock 1:1 with dH₂0), incubate all in 95°C block for 10 min, spin 1 min at 14K (RT) then place tubes on magnetic separation rack and transfer supernatant to new tubes (minimize risk of loading beads onto gel). Then load gel or freeze samples at -20°C.

ƛ-phosphatase Treatment Protocol

14. After final wash (step 11) 

15. Once beads are re-suspended in the 2^nd wash of IP buffer + inhibitors, split samples you want to treat with phosphatase. Using a cut pipette tip, mix suspension thoroughly and quickly transfer ~131 µL to fresh, pre-chilled tubes. 

16. Each sample being treated for phosphatase should have 2 tubes now containing roughly half of the IP beads in each. Label one for ƛ-phosphatase treatment and one for mock treatment. Spin these samples down as before and remove supernatant off beads. 

17. During washes, prepare phosphatase and mock treatment solutions. The volumes below are for a single sample (half of an IP so roughly 2.5µL of IP beads). Calculate Master Mixes accordingly.

1X PPase reaction                           1X Mock reaction

H₂O                                                 2.5 µL                                                  3.5 µL            

Ppase buffer (10X)                          2 µL                                                      2 µL

10mM MnCl2                                   2 µL                                                      2 µL

l-ppase                                            1 µL                                                                 

Total                                                 7.5 µL                                                 7.5 µL

18. Remove final wash solution from beads and re-suspend in 7.5 µL of either phosphatase or mock treatment solution.

Protein A bead stock used for IP’s should be a 1:1 solution of beads in storage solution. Using 10 µL of protein A slurry for each IP would be 5 µL of actual beads for each IP. If you split the sample during the final wash, you will then have 2.5 µL of beads per IP sample for ppase or mock treatment. 

19. Incubate tubes at 30°C for 30 min in pre-set Eppendorf Thermomixer at 500rpm.

20. Let stand in NEB magnetic stand and remove phosphatase and mock treatment solutions. Add 20 µL of 1X SDS loading buffer to treated IP samples, incubate at 95°C for 10 min, spin down at 14k for 1min, then let tubes stand in NEB magnetic stand. Finally, transfer supernatant (This should contain the denatured IP’d proteins + protein A from the beads) to fresh tubes and then load gel or freeze these samples at -20°C.