TAP Fusion Protein Purification from Yeast
A Dohlman Lab Protocol
1. Inoculate single colony into 5 mL appropriate selective media and grow overnight.
2. Using above culture inoculate a new culture >1:50 into the 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 4oC 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 sec and aspirate off supernatant.
6. Add 1 mL 1 mM NaAz and measure OD600 using 5 uL 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 uL 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. (Scary spin = poke a hole in the bottom of the tube, and spin out the liquid into a second 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 40 uL 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 uL of lysis buffer + PIC + 1 mM CaCl2 to the tube, vortex and spin for 20 sec at 3000 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 (500 uL) of cleared lysate to new tubes.
19. Remove 30 uL cell lysate and put aside for SDS-PAGE analysis of whole cell extract.
20. Add 250 mM CaCl2 to final concentration of 1 mM to each reaction (for 500 mL, use 2 mL CaCl2). Alternatively, the CaCl2 can be added to the lysis buffer in the beginning rather than at this stage.
21. Add 40 uL washed beads to each reaction.
22. Rock samples for 2 hrs at 4oC.
23. Spin down samples for 20 sec at 3000 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 1 mL Lysis buffer + 1 mM CaCl2 and transfer to a new tube.
25. Mix for 2 min at 4oC.
26. Spin down samples for 20 sec at 3000 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 50 uL 2X SDS Buffer and incubate at 65oC for 10 min, 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 40 uL 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 1 M Na2HPO4
8.3 mL 1 M NaH2PO4
-400 mM NaCl
80.0 mL 5 M NaCl
-10 % glycerol
123.3 g 100% glycerol (@ 25 ˚C)
-0.1% Triton X-100
-25 mM NaF
-25 mM glycerophosphate
Prior to use, supplement 49 mL of 1X IP Lysis Buffer with:
-50 mM NaVO3
-0.5 M DTT (77 mg/mL)
50 uL. [FRESHLY PREPARED]
-PIC = 1 tablet protease inhibitor cocktail (e.g. CompleteTM EDTA-free Protease inhibitor cocktail tablets; Roche Diagnostics; Catalog #1 873 580).