General Protein Purification Protocol
Edited by Andy McMillan 1-15-2015
A. Weigh cell pellets in bottles before resuspension (and the bottles after removing
resuspended pellets).
B. *** resuspension volume depends on lysis method:
For sonication:
Resuspend Cells in 40 ml Loading buffer
Recommended: save an aliquot (equivalent to 5ml culture) for miniprep and
sequencing to verify the identity of the protein (good idea if you’re looking at
lots of mutants)
add 80 l PMSF and 400 l DNase (~50X stock). Incubate on ice for 1 hour.
Sonicate Cells (settings: 50% Amplitude, 1s on/1s off pulse, 20 minutes.
Larger pellets may need to be split and/or require additional time after
replacing ice)
Resuspend in smaller volume for bead beater or microfluidizer
C. Pellet the lysate – Oak ridge round bottom tubes (~45 ml), SS-34 rotor, 15,000
RPM, 30’, 4o
D. Filter Supernatant with 0.2 m filter (50 ml steriflip is easiest)
E. Set up FPLC (while cells are spinning)
1. Make sure there is enough filtered ddH2O, 20% EtOH, and buffers A & B
buffer A (loading/binding buffer):
buffer B (elution buffer):
2. Remove air from inlet tubing
3. PumpWash A & B w/ ddH2O
4. Also wash out tubing w/ 1-2 ml ddH2O (in load and inject positions)
5. Wash Column w/ ddH2O to remove ethanol (5 column volumes (CV); in load
position)
6. Superloop should have been stored assembled in 20% EtOH: wash Superloop
with 4 Vol MilliQ water using 60 ml syringe if it has been stored assembled with
20% EtOH)
7. PumpWash A & B with binding buffer (A) and elution buffer (B)
8. Connect inlet of superloop to position 6; fill 60 ml syringe w/ filtered sample and
inject into superloop; Connect superloop to position 2 (Do this with FPLC set on
Load; water in superloop will go to waste when sample is injected)
9. Run program:
10. Collect fractions containing target protein. Save samples to run on gel (G).
F. Clean FPLC
1. Move pump intakes to water; do PumpWash
2. Wash column w/ water – 5 CV (25 ml) @ 3 ml/min
3. Move pump intakes to 20% EtOH; do PumpWash
4. Wash column w/ 20% EtOH for 5 VC (25 ml) @ 3 ml/min; Remove column and
cap and parafilm ends; label w/ protein and date and store @ 4 o
5. detach superloop, disssemble by removing the inner and out end pieces from one
end; use a syringe filled with water to push the inner moveable seal out; wash
with MilliQ water
6. do final wash  PumpWash; Load Wash and Inject Wash (2-5 ml each)
7. Empty waste containers
G. Run samples on gel (10% Bis Tris)
Lysate and pellet:
2 l sample
18 l binding buffer A or water
20 l 2X Loading dye
4 l 1 M DTT
Other Fractions:
20 l sample
20 l 2X Loading dye
4 l 1 M DTT
Heat at 70º for 10’
Run gel 200V, 50’
H. Pool fractions
1. Pool fractions containing protein. Concentrate and/or exchange into storage
buffer.
2. Measure OD280 to get protein concentration.
3. Add glycerol to a final concentration of ~25-30%.
4. Aliquot protein into eppendorf tubes (50-100 ul per tube, or as needed)
5. Freeze in -80.