UBA1 Protocol
Updated: 2/17/12
Expression and Purification of UBA1 (E1 from wheat germ)
MW = 119 kD
ε280 = 91.3 cm-1mM-1
pI ~ 5.16
Note: This construct contains an N-terminal His tag
BUFFERS
Lysis Buffer
o 50 mM Tris-HCl pH 8.0
o 1 mM EDTA
o 1 mM DTT
Equilibration Buffer
o 50 mM Tris HCl pH 8.0
Wash Buffer
o 50 mM Tris-HCl pH 8.0
o 0.5 M KCl
Elution Buffer
o 50 mM Tris-HCl pH 8.0
o 10 mM DTT
Regeneration Buffer
o 50 mM Tris-HCl pH 9.0
o 1.0 M KCl
Storage Buffer
o 50 mM Tris, HEPES, or MOPS pH 7-7.5 + 0.2% Azide.
Growth:
1. Grows cells at 37°C in 6 x 1L LB containing kanamycin (50 ug/mL working
concentration).
2. Induce cells with IPTG (0.1 mM final concentration; ~25.0 mg/mL) when OD600
reaches 0.7-0.8 and let express at 16°C overnight.
a. Take a pre-induction sample for SDS-PAGE analysis.
3. Harvest cells the following morning by centrifugation at 5000 rpm for 20
minutes.
4. Resuspend cell pellet in Lysis Buffer:
a. 50 mM Tris-HCl pH 8.0
b. 1 mM EDTA
c. 1 mM DTT
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UBA1 Protocol
Updated: 2/17/12
Protein Purification:
1. Add His-protease inhibitor cocktail to thawed cells.
2. Add MgCl2 (to ~10 mM from 1M stock) to the cells and a small scoop of solid
DNase and RNase.
3. Run cell suspension twice through the French Press while making sure to add
50 uL of 250 mM PMSF (43mg PMSF per 1 mL EtOH) after each press.
a. Upon completion of lysis, check viscosity of lysate via pipetting to
confirm complete digestion of DNA/RNA; let stand on ice for awhile
longer if sample is even slightly viscous.
4. Clarify the lysate via centrifugation at 17500 rpm in SS34 tubes for 20 minutes.
a. Take a post-induction sample for SDS-PAGE analysis.
5. Remove supernatant and place it into a beaker and adjust the pH to 7.5-7.8
with 1M NaOH.
6. Add fresh ATP (0.1M Stock) to 2mM; Phosphocreatine to 6mM (0.1M Stock);
Add a small amount of solid Phosphocreatine Kinase (PCK)
a. May also be labeled as Creatine Phosphokinase.
Preparing Stocks:
I. ATP (81 mg in 1.5 mL Equilibration Buffer = 0.1M) pH to ~7 with Na2CO3.
Keep on ice.
i. Adding a small scoop of Na2CO3 will raise the pH. The sol’n will
eventually buffer at 7; verify pH with pH paper.
II. Phosphocreatine (31.61mg in 1.5mL = 0.1M) No need to pH.
i. Make ~4.8 mL PCr for ~80 mL Lysate!
Addition to Lysate:
I. ATP: 400 uL of 0.1M to 20mL Lysate = 2mM (MW = 551 g/mol)
II. Phosphocreatine: 4.8 mL of 0.1M to 80mL Lysate = 6 mM (MW = 255
g/mol)
7. Wash Ub-AffiGEL-10 resin with equilibration buffer. Mix resin (~5 mL) with
extract either in 50 mL conical or in BioRAD Econo Column.
a. NEVER LET THE COLUMN DRY OUT!
8. Seal ends and nutate at room temperature for ~30+ minutes.
9. Clamp column to ringstand in the cold-room and allow the extract to flow
through.
a. Collect a sample for SDS-PAGE
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UBA1 Protocol
Updated: 2/17/12
10. Wash the column with 20+ volumes of Wash Buffer.
a. Collect a sample for SDS-PAGE
b. Check A260 and A280 before adding elution buffer.
i. Note: Make sure that A260 reading is close to 0! Blank with wash
buffer. There is a lot of DNA in 6L worth of cells, so we need to be
sure to get rid of it all! It’ll take about 100 mL to remove all DNA if
you add wash buffer in small amounts to the column, letting it
flow through, and then adding more.
11. Seal the column, add ~5 mL of Elution Buffer and let sit for 10 minutes prior to
eluting UBA1.
12. Add another 5 mL of elution buffer and let it flow through to collection conical
to collect more UBA1.
a. Repeat previous step 3 more times.
13. Seal the column and add a final ~5 mL of Elution Buffer. Let this sit for 10 more
minutes prior to eluting the residual UBA1 from the column.
14. Elute UBA1.
a. Collect a sample for SDS-PAGE.
15. Dialyze UBA1 into 50 mM Tris, 1mM DDT pH 7.5 or 50 mM HEPES, 1mM DTT
pH 7.5.
a. HEPES might be the better option here since makes a better buffer at
this pH.
16. Run a gel on each collected fraction and determine relative protein
purity/amounts.
17. Spec the post-dialysis sample to determine protein concentration.
18. Concentrate UBA down to either 10 uM or 40 uM (depending on what the lab
needs).
a. Be sure to consider the future addition of 5% Glycerol and 1 mM DTT
when performing your concentration calcuations.
19. Add glycerol to 5% and 1mM DTT. UBA1 concentration should NOW be at
either 10 uM or 40 uM.
a. Glycerol and DTT should not dilute sample beyond this if you took these
into account during your concentration calculations.
20. Divide sample into 100 uL aliquots and flash freeze and store at -80C.
21. Regenerate Ub-AffiGEL column by washing with regeneration buffer.
22. Store column at 4°C in storage buffer.
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