A Dohlman Lab Protocol

Cell Lysis and Batch Affinity Purification

1. Inoculate single colony into 5 mL appropriate selective media and grow overnight.
2. Using above culture reinoculate >1:50 into 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 seconds 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 400 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. We use a vortex fitted with a 60-microtube headpiece or a MixMate to vortex multiple tubes simultaneously.
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 30 min to further solubilize proteins. (Optional)
14. Carefully mix the FLAG resin 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 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. Alternatively use a compact reaction column (USB Corp.) and vacuum.
17. Spin lysates at max speed for 1 min at 4oC to clear lysate and transfer supernatant to new tube. Repeat for 30 min.
18. Transfer cleared lysate to new tubes.
19. Remove 30 uL cell lysate and put aside for SDS-PAGE analysis of whole cell extract, and remove 5 uL for protein concentration assay.
20. Equalize protein concentration by diluting with lysis buffer, then mix with FLAG-resin
21. Rock samples for 2 hrs at 4oC.
22. 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.
23. Add 500 uL lysis buffer and transfer to a new tube.
24. Mix for 2 min at 4oC.
25. 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.
26. Resuspend the bead pellet in 50 uL 2X SDS Buffer and incubate at 65oC for 10 min, occasionally mixing tube. Alternatively elute bound protein twice with 20 uL of “3xFLAG” peptide (Sigma, 0.25 mg/ml final concentration), mix with 10x SDS-PAGE sample buffer.
27. 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.
Remove 40 uL of supernatant, careful not to remove any beads. Now ready to run on SDS-PAGE.

Stock FLAG Lysis Buffer (50 mL)

10 mM Tris-HCl pH 8.0
200 mM NaCl
0.1% Triton X-100
25 mM beta-glycerophosphoate
1 mM EDTA
PIC=2 tablets protease inhibitor cocktail per 50 mL (e.g. CompleteTM EDTA-free Protease inhibitor cocktail tablets; Roche Diagnostics; Catalog #1 873 580).

Original FLAG fusion protein purification from Yeast optimized for mass spec sequencing

Cell Lysis and Batch Affinity Purification

1. Begin by making 50 mL of fresh lysis buffer by adding protease and phosphatase inhibitors to an aliquot of the pre-chilled lysis buffer (see recipe below).
2. While the lysis buffer is mixing, fill the appropriate number of microfuge tubes with 400 ul of clean Biospec 0.5 mm glass beads and keep tubes chilled on ice (note: you’ll need approximately as many tubes as you have milliliters of cell suspension).
3. Once lysis buffer is well mixed, add 1 to 2 cell-pellet-equivalent volumes of lysis buffer to the cell pellet. Ensure the pellet is well mixed with the buffer by vigorously shaking and swirling. Mix to homogeneity by shaking.
4. Add 750 uL of the cell/buffer mixture to the pre-chilled microfuge tubes that contain glass beads and close each tube.
5. Lyse the cells by vortexing the microfuge tubes at the maximum setting for at least 30 min at 4°C (or until you’ve achieved >75% lysis as determined by checking a small aliquot of the cell mixture by light microscopy). We use a vortex fitted with a 60-microtube headpiece to vortex multiple tubes simultaneously.
6. After lysis is complete, spin the insoluble material down for 15 min at max speed (13200 rpm/16.1 rcf) in a microcentrifuge chilled to 4°C.
7. After pelleting the crude lysate, transfer the cleared lysate to a chilled 50 mL conical tube (on ice) making sure not to disturb the pellet or the glass beads.
8. Optional: Spin the pooled lysate for 1 hour at 4°C, 25000 rpm in pre-chilled SW-28 rotor.
9. During centrifugation, equilibrate the M2-FLAG affinity resin (SIGMA, F2426-1ML) with lysis buffer twice with 10 min incubations at 4°C (We typically use ~200 uL bead volume (400 uL 50% slurry) for a 9-OD600 cell pellet (i.e. 10 L prep grown to OD600 = 0.9) in a ~250 mL lysis buffer volume. For a 50 mL lysate, we would use no less than 50 uL of bead volume.
10. Mix the equilibrated M2-FLAG affinity resin into the cleared lysate and incubate on a rotisserie at 4°C for 2 – 3 hours.
11. Harvest the affinity resin by spinning down the resin-lysate mixture at 4°C, 3000 rpm table top centrifuge, for 5 minutes. Carefully remove the supernatant, making sure not to disturb the pelleted affinity resin.
12. Wash the resin 3 x 10 mL x 10 min with lysis buffer. Then transfer the resin to a 1.5 mL microfuge tube and wash once more with 1 mL lysis buffer (spin at 9000 rpm microfuge = 7.5 rcf).
13. Elute the FLAG-tagged protein from the affinity resin by incubating the resin at 30°C for 15 minutes in lysis buffer containing 0.25 mg/mL “3xFLAG” peptide. Shake at 950 rpm.
14. Spin down sample (9000 rpm, 2 min) and transfer eluate to fresh 1.5 mL Axygen tube. Repeat step 13 and collect eluate into same tube as first.
15. Spin down eluate tube and again transfer eluate to fresh 1.5 mL tube MAKING SURE NOT TO TRANSFER ANY BEADS, WHICH WILL CAUSE PROBLEMS WITH DOWNSTREAM ASSAYS!!! (One may opt ot use a compact reaction column to trap the affinity resin during elution).
16. Separate the eluate by SDS-PAGE followed by in-gel digestion for MS analysis.

Lysis Buffer

Premade Buffer to sit @ 4°C Stock @ Add to 1L
25 mM HEPES-NaOH pH 7.5 238.3 g/mole 5.97 g
400 mM NaCl 58.44 g/mole 23.376 g
10% Glycerol 100% 100 mL
0.5 mM DTT 1 M DTT 0.5 mL
0.1% Triton X-100 100% 1 mL

Add immediately prior to use: Stock @ Add to 50 mL
25 mM NaF 0.5 M 2.5 mL
1.3 mM activated Na-O-vanadate 200 mM 0.325 mL
50 mM Beta-glycerophosphate 216 g/mole 0.54 g
Complete Protease Inhibitor Tablets 1/50 mL 1
0.5 mM PMSF 100 mM 0.25 mL
Lysis buffer – 47 mL

Reference:

Hall MC, Torres MP, Schroeder GK, Borchers CH. (2003) Mnd2 and Swm1 are core subunits of the Saccharomyces cerevisiae anaphase-promoting complex. Jour. Biol. Chem. 278 (19): 16698 – 16705.