1. Grow several liters of cells overnight to the density 2-5×10e7 cells/ml
2. Spin down cells in 1 liter bottles at 5000rpm 15-20’ 4ºC
3. Remove supernatant and collect cells with up to 50ml of ice cold water into 50ml conical tube
4. Spin down cells for 1’ at 4000rcf and remove the supernatant.
5. Add 0.5-1ml of IP buffer(see the recipe below) containing protease inhibitor cocktail. Use a spatula to re-suspend
6. Slowly pour the cells into a new 50 ml tube filled with liquid nitrogen and emerged in it. The slower the procedure, the smaller size of the frozen pellets, and lesser chance of possible sudden boiling of liquid nitrogen.
7. Important: Make several holes in a cap with a razor blade to exhaust nitrogen. This step is necessary to avoid explosion of the tubes because of the high pressure of the nitrogen vapors.
8. Pour off excess liquid nitrogen through the holes (use protective glasses and insulating gloves or forceps). Cells can be maintained at –80ºC for a long time at this stage.
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9. Put pellets in the screw-top jars previously immersed in the liquid nitrogen until it stops boiling. Insert the jars into a Retsch Mixer Mill MM301, and beat for 5 times, 3’ at 30 beats per second. Between each beating immerse the jars in liquid nitrogen to cool. Efficiency of cell breakage can be observed in a microscope.
10. Transfer a cell powder back into 50 ml tube pre-cooled in liquid nitrogen. Be sure that there is no residual liquid nitrogen left in the tube. Cell powder can be maintained at –80ºC for long time at this stage.
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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 and cool before runs.
13. Solubilization step: incubate rotating 45’-60’ @ 4ºC
14. Spin down at full speed 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 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! Its 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.