A Dohlman Lab Protocol

FLAG Fusion Protein Purification from Yeast

 
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 500 ul of clean Biospec 0.5mm glass beads and keep tubes chilled on ice (note:  you’ll need as many tubes as you have milliliters of lysate).  
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 1 mL 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 genie fitted with a 60-microtube headpiece.  
6. After lysis is complete, spin the insoluble material down for 15 minutes at max speed (13.2 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 Ultracentrifugation) Spin the 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 minute incubations at 4°C (We typically use ~200 uL bead volume (400 uL 50% slurry) for a 9-OD600 cell pellet (i.e. 10L prep grown to OD600 = 0.9) in a ~250 mL lysis buffer volume.  For a 50 mL lysate, we would use no less than 50uL 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 50 mL extract at 4°C, 5000 rpm, for 5 minutes.  Carefully remove the lysate, making sure not to disturb the pelleted affinity resin.
12. Wash the resin 3 x 25 mL x 10 min with lysis buffer.  Then transfer the resin to a 1.5 mL microfuge tube and wash once more with 1mL lysis buffer (spin at 9000 rpm / 7.5 rcf).
13. Elute the FLAG-tagged protein from the affinity resin by incubating the beads at 30°C for 15 minutes in lysis buffer containing 0.5 mg/mL 3x FLAG peptide.  Shake at 950 rpm.
14. Spin down sample (9K, 2 minutes) 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    5.97g
400 mM NaCl                                    58.44 g/mole    23.376g
10% Glycerol                                     100%                100mL
0.5 mM DTT                                      1M DTT           0.5mL
0.1% Triton X-100                             100%                1mL
        
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.  


            

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