1. Culture 20 flasks (Corning, 430641; 75 cm2 culture area/flask) of mammalian cells until reach over 95% confluent.
2. Remove the culture medium, and rinse the cells twice with cold 1X PBS and aspirate.
3. For each flask, add 1 ml IP Buffer (20 mM Hepes (4.79 g/L), 2 mM MgCl2 (0.19 g/L) and 250 mM NaCl (14.5 g/L), PH7.4) + 0.05% Tween 20 + protease inhibitors, and rinse the cells.
4. Freeze the cells (still attached on the bottom of the flask) with liquid nitrogen, keep them at -80℃ or
5. Thaw them at 4℃, and tap the flask to detach the cells from the bottom, and collect the cells of all flasks into a 50 ml tube.
6. Spin down the cells at 3000g, and transfer the supernatant to a new 50 ml tube (don’t discard it!).
7. Add 1 ml the supernatant to the cell pellet (~ 3-5g), and vortex it to suspend the cells.
8. Get a new 50 ml tube filled with liquid nitrogen, and drip the suspended cells into the liquid nitrogen to get frozen cell pellets (diameter ~3-5mm).
9. Break the cells as described for cryogenic breaking yeast cell.
10. Put back the supernatant (from step 6) to the broken cells, do IP as for the protein from yeast cell.

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11. For 1 IP experiment: Re-suspend 1-5 g of broken cells in 5-10ml IP buffer +0.1% Tween 20 +Cocktail of protease inhibitors + kinase inhibitors.
12. Let the tube thaw on ice for 10-15 min and then use a tissue homogenizer to mix. Mix quickly, for 15-30 seconds/one run, and cool before runs (2-4 runs).
13. Solubilization step: incubate rotating 30’-60’ @ 4ºC
14. Spin down at full speed at 3500-5000 rm in the low speed centrifuge for 1’-5’ @ 4ºC
15. Transfer sup into new 10ml tube/s
16. Add 5-10 mg of Dynal beads with immobilized antibody to each sample.
17. Rotate 15-30’ 4ºC
18. Collect the beads using a big magnet. Discard all sup except for 1ml – transfer it to a new 2ml eppendorf tube.
19. Wash 3X with IP buffer. Change to a new 1.65 ml tube.
20. Add 200µl 3XFLAG (from stock of 200µg/ml in 50mM Tris-HCl pH=7.4, 150mM NaCl))
21. Elute rotating for 15-30’ @ 4ºC
22. Transfer 200µl of supernatant to new 1.65 ml tube.
23. Add 1000µl IP buffer + 20µl Cobalt Talon beads from a commercial stock.
24. Rotate 4ºC 15’
25. Remove sup
26. Wash 2X1ml IP buffer, change the tube. Use few seconds spin to remove residual amount of washing liquid.

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Separate sample onto two parts by separating the liquid containing the Talon beads.
For separating the purified proteins by SDS-PAGE process as follows:
NOTE – FROM NOW ON BE VERY CAREFUL TO AVOID CONTAMINATING YOUR SAMPLE WITH KERATINS
27. Add 10µl Sample buffer containing 200mM Imidazole without reducing agent! It presence rusts the Co beads.
28. Incubate 15’ 37ºC rotating
29. collect the sup using magnets
30. Add reducing agent and reduce for 15-30’ at 60 C.
31. Run SDS-PAGE use Micro-Gel 15’ or any suitable gel electrophoresis.
FOR ON BEADS DIGESTION
27a. Wash the remainig Co-Talon beads two times with 1 ml of 50mM ammonium bicarbonate buffer. And change the tube.
28a. Add 10 ul of 1pmol’ul trypsin solution in 50 mM ambic to the beads. Incubate 5-30 min; collect the sup and incubate further for 5-12 hours. Limited digestion may help to minimize elution of nonspecifically bound proteins (?, remains to be proven) Another option is just add trypsin and digest on the beads all time (6-12 hours)
29. Analyze 1/3 or ½ of the digest directly by MALDI or HPLC-ESI.
30. For HPLC-ESI, spin down the sample using high speed centrifuge to minimize the chance of blocking the tubes of an HPLC system.