Ube2t Purification Protocol (15N MOPS Prep)
2-DAY PREP
HIS-TAG REMOVAL IN THIS PREP
Growth and Expression
1. Transform His-Ube2t into the BL21 (DE3) Star cell line.
2. Plate cells overnight on the appropriate antibiotic plate (Ampicillin).
3. The next day, inoculate MOPS media with cells from the plate and add the
appropriate antibiotic (Ampicillin).
4. Incubate cell culture at 37C until and OD600 of ~0.6 is reached.
5. Add IPTG to 0.5 mM and incubate overnight for ~22 hours.
6. Harvest cells in Ni2+ start buffer, pH 7.6.
7. Store at -80C until you are ready to purify.
Purification
1.
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6.
Remove cells from freezer and thaw at room temperature.
Add 1 aliquot of His-protease inhibitor cocktail.
Add small amounts of DNase + RNase.
Add MgCl2 to 10mM.
Add 60 uL of saturated PMSF (~43 mg/mL).
French press cells twice; make sure to add 60 uL of saturated PMSF after each
press.
7. Centrifuge cells in an SS34 tube at 15,000 rpm @ 4C.
8. While cells are spinning in the centrifuge, being equilibrating a Ni2+ column with
nickel start buffer.
9. Harvest the supernatant and pellet when done spinning and take samples for an
SDS-PAGE gel.
10. Add some His-PI and some saturated PMSF to supernatant.
a. Filter through a 0.22 um low protein-binding syringe filter to remove
anything that has crashed out.
11. Pour supernatant over the nickel column and collect the FT for SDS-PAGE.
a. Take a sample for SDS-PAGE.
12. Wash the column with 2 column volumes (15 mL) of 50 mM imidazole solution.
a. Collect sample for SDS-PAGE.
13. Begin eluting protein with 1CV (15 mL) 250 mM imidazole solution.
a. Collect a sample for SDS-PAGE.
i. Ube2t should be primarily in this fraction!
14. Continue elution with 500 mM Imidazole.
a. Collect a sample for SDS-PAGE
15. Add some His-PI and PMSF to each fraction and store @ 4C.
16. Run a 15% SDS-PAGE gel on all collected samples.
17. After the gel is finished, check to see where Ube2t has eluted off.
a. It should be in the 250 mM Imidazole elution fraction.
18. Pool Ube2t-positie fractions into a dialysis bag and dialyze at room temperature
for 3 hours in 4 L of 25 mM Tris, pH 7.0 w/ 2-3 aliquots of TEV-protease.
a. Take a “Pre-TEV” sample for SDS-PAGE.
19. After 3 hours, transfer to the cold room (4C) and continue dialysis overnight.
20. The next morning, remove sample from dialysis and run over a clean and freshly
equilibrated (in dialysis buffer) Nickel Column.
21. Collect the FT.
a. Collect a sample for SDS-PAGE
22. Wash the column with 1 CV (15 mL) 50 mM Imidazole and collect elution.
a. In the past, Ube2t has eluted off primarily in this fraction!
b. Collect a sample for SDS-PAGE
23. Elute the remaining protein with 1 CV (15 mL) 250 mM Imidazole.
a. Collect a sample for SDS-PAGE.
24. Elute any residual protein with 1 CV (15 mL) 500 mM Imidazole.
a. Collect a sample for SDS-PAGE.
25. Run a 15% SDS-PAGE gel on resulting fractions to determine location of Ube2t.
26. Pool (if necessary) Ube2t-positive fractions and add some Non-His PI (contains
EDTA).
27. Store at 4C overnight if necessary; if it is possible, try and finish the purification
today.
a. Very possible if you work efficiently.
28. Concentrate for SEC chromatography in 10K MWCO concentrator (~1.5 mL final
volume)
29. Run concentration sample over the SEC column and collect Ube2t fractions.
30. Run an SDS-PAGE gel on SEC fractions and look for degradation products.
a. Compare fractions pre and post-SEC.
b. NOTE: You might see ‘breakdown’ products on your gel. This may be the
disordered C-terminus getting cleaved at some point during the
purification. Try and minimize pooling fractions with a significant amount
of this ‘breakdown’, if possible. In the past, minor breakdown products
have not resulted in labeled preps yielding bad spectra. In other words,
don’t worry about it too much, so long as your final pool isn’t mostly
degraded.
31. Pool all good-looking fractions and concentrate to appropriate concentration.
32. Flash freeze in liquid nitrogen and store at -80C.