Ubiquitin Expression and Purification
Updated: 8/22/11
wt-Ubiquitin Expression and Purification
MW = 8564.8 kDa
ε280 = 1.42 cm-1mM-1
HA-Ubiquitin ε280 = 5.96 cm-1mM-1
pI ~6.56
ZY Media Prep (Pre-autoclave)
 10g Tryptone
 5g Yeast Extract
ZY Media Prep (Post-autoclave)
 Add 1 mL of 1M MgSO4 stock
 Add 20 mL of 50X 5052
 Add 50 mL 20X NPS
Growth:
1. Grow cells overnight at 37C in 1L of auto-induction “ZY” media + Ampicillin
(100 ug/L).
2. Harvest cells the following morning by centrifugation at 5000 rpm for 20
minutes.
3. Resuspend cells in lysis buffer.
a. 50 mM Tris-Hcl, 1 mM EDTA, 0.05% Triton X-100 pH 7.6
Protein Purification: [DAY 1]
1. Add ∆His-tag protease inhibitors to the thawed suspension of cells.
2. French press cells twice through the French Press
a. Add 50 uL PMSF after each press (43 mg PMSF/1 mL EtOH).
3. Add MgCl2 to ~5 mM and small amounts of DNase and RNase to the cell
extract; mix by inversion and let stand on ice for 30+ minutes.
4. Clarify lysate at 12,000 rpm for 30 minutes in an SS34 rotor.
5. Transfer supernatant to a beaker (on ice) with a stir bar in it.
6. Slowly (over a period of ~2-3 minutes) add 70% perchloric acid until pH drops
to 4.5 (should be ~0.35 mL acid/50 mL Lysate) while stirring. Add the acid in a
“star pattern” in order to avoid the “local pH” dropping too far.
a. The solution will turn milky; continue stirring on ice for about 30
minutes following addition of all of the perchloric acid.
b. Check to make sure that pH is 4.5 with pH paper.
7. Centrifuge suspension at 18,000 rpm for 20 minutes in an SS34 rotor.
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Ubiquitin Expression and Purification
Updated: 8/22/11
8. Dialyze the supernatant against cold 50 mM Sodium Acetate, pH 4.5 overnight.
a. NOTEAdjust the pH with glacial acetic acid, not HCl. avoid adding salts
since we are performing ion exchange chromatography for the secondary
purification step.
[DAY 2]
9. Dialyze again the following morning in 50 mM Sodium Acetate, pH 4.5 for 3-4
hours.
a. Again, be sure to adjust the pH with glacial acetic acid.
10. Apply the supernatant to a 15 mL SP column pre-equilibrated with 50 mM
Sodium Acetate pH 4.5.
a. Allow supernatant to flow through and collect a sample for SDS-PAGE
analysis.
11. Wash the column by directly adding 1-2 column volumes of 50 mM Sodium
Acetate, pH 4.5.
a. Collect the wash and take a sample for SDS-PAGE analysis.
12. Elute protein with a 200 mL linear gradient from 0-0.5 M NaCl in 50 mM
Sodium Acetate pH 4.5.
a. Ubiquitin usually elutes around the half-way point of the gradient.
13. Check for protein containing fractions via UV absorbance.
a. Collect samples from protein fractions for SDS-PAGE analysis.
14. Run an SDS-PAGE gel on protein fractions to determine purity.
a. Pool pure ubiquitin containing fractions.
15. Dialyze ubiquitin into a convenient buffer for experimental applications.
a. 25 mM NaPO4, 150 mM NaCl pH 7
b. 25 mM HEPES pH 7.5
c. 25 mM Tris-HCl pH 7.6
16. Concentrate to 3 mM and divide into 100 uL aliquots.
17. Store at -20C (no need to flash freeze).
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