1. Inoculate single colony into 5 mL appropriate selective media and grow overnight.
2. Using above culture inoculate a new culture 1:30 into desired amount of appropriate media in the morning.
3. That afternoon, cultures should be at OD600=0.9-1.0. Add sodium azide (NaAz) to final concentration of 10 mM. Spin down that culture for 5 min at 4000 x g at 4oC.
4. Keep everything at 4o C from this point forward!
5. Resuspend the pellet in 1 mL 10 mM NaAz and transfer to a microfuge tube. Spin at max speed for 15 seconds and aspirate off supernatant.
6. Add 1mL 1mM NaAz and measure OD600 using 5uL of culture.
7. Transfer desired amount of cells (x) to a new tube to equalize cell volumes: 
8. Spin at max speed for 15 sec and aspirate off supernatant.
9. At this point the cell pellet can be used now or be frozen at -80oC for use later. When ready to use add lysis buffer (prevents degradation during thaw) to frozen pellet and thaw.
10. Resuspend pellet in at least 650 mL lysis buffer + PIC and add a scoop of glass beads.
11. Lyse cells by vortexing at max speed for 10 x 30 sec, keeping cells on ice in-between.
12. Scary spin the cell lysate into a new tube.
13. Mix lysates at 4oC for 1 hr. to solubilize proteins.
14. Carefully mix the Calmodulin Gel beads until completely and uniformly suspended.
15. Aliquot out 40mL of the slurry per reaction into a microfuge tube. Note: Cut the end of the pipette tip off to dispense slurry easily.
16. Wash the beads by adding 500 mL of lysis buffer + PIC + 1mM CaCl2 to the tube, vortex and spin for 20 sec at 3K RPM. Carefully remove the supernatant. Repeat 4 times and place tube with washed beads on ice.
17. Spin lysates at max speed for 1 min at 4oC to clear lysate and transfer supernatant to new tube. Repeat for 15 min.
18. Transfer equal amount (500mL works good) of cleared lysate to new tubes.
19. Remove 30 mL cell lysate and put aside for analysis of whole cell extract.
20. Add 250mM CaCl2 to final concentration of 1mM to each reaction (for 500mL, use 2mL CaCl2). Alternatively, the CaCl2 can be added to the lysis buffer in the beginning rather than at this stage.
21. Add 40mL washed beads to each reaction.
22. Rock samples for 2 hrs at 4oC.
23. Spin down samples for 20 sec at 3K RPM, allow tubes to sit for 30 sec so beads settle to the bottom of the tube (VERY IMPORTANT because these beads tend to stick to the side of the tube); carefully aspirate off supernatant.
24. Add 1mL Lysis buffer + 1mM CaCl2 and transfer to a new tube.
25. Mix for 2 minutes at 4oC.
26. Spin down samples for 20 sec at 3K RPM, allow tubes to sit for 30 sec so beads settle to the bottom of the tube; carefully aspirate off supernatant. Repeat 4 times.
27. Resuspend the bead pellet in 50mL 2X SDS Buffer and incubate at 65oC for 10 minutes, occasionally mixing tube.
28. Spin down samples for 20 sec at 3K RPM, allow tubes to sit for 30 sec so beads settle to the bottom of the tube.
Remove 40mL of supernatant, careful not to remove any beads. Now ready to run on gel.

Stock 1X IP Lysis Buffer (1.0 L)

-50 mM Sodium Phosphate Buffer pH 7.5    
        41.7  mL 1M Na2HPO4
        8.3  mL 1M NaH2PO4

-400 mM NaCl                    
        80.0  mL 5M NaCl

-10 % glycerol                         
        123.3  g 100% glycerol (@ 25 ˚C)

-0.1% Triton X-100                 
        1.0  mL

-25 mM NaF                     
        1.05 g

-25 mM glycerophosphate             
        5.4   g

Prior to use, supplement 49 mL of 1X IP Lysis Buffer with:

-50 mM NaVO3                     
        1 mL 

-0.5 M DTT (77 mg/mL)                 
        50 uL.  [FRESHLY PREPARED]

-1 tablet  protease inhibitor cocktail (e.g. CompleteTM EDTA-free Protease inhibitor cocktail tablets; Roche Diagnostics; Catalog #1 873 580).