32P
labeled GST Ubiquitin
1. pGEX-2TK-Ub plasmid is in BL21(DE3). The 10 ml LB (Amp 150 mg/l)
over night preculture was added to 150 ml LB (Amp 150 mg/l) and continue
to shake to 0.6 (OD600). Collect cells and resuspended in 4 ml lyses buffer
(1%triton X-100, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, add PMSF at 1
mM (1:200 stock in methanol store at 4C) and 0.8 mg DTT/ml. Sonicated 3 x
12 Sec. on ice. Scale between 3 and 4 of ?). Centrifuge 11,000 rpm x 20 min.
and take supernatant and check proteins.
2. Binding to GSB. Add 100 l pre-washed glutathione beads to 750 l crude
extract and bind at RT for 30-60 min. Wash with 3 x PBS. Wash 3 x with 1 x
kinase buffer (10 x kinase buffer 200 mM Tris pH 7.5, 1 M NaCl, 120 mM
MgCl2, 10 mM DTT). Remove final wash by very thin tip.
3. Labeled reaction.
Add 5 ul 10 x kinase buffer
1 ul protein kinase*
2 ul gamma 32P-ATP (Amersham #AA0018)
42 ul d H2O
Incubate the reaction on ice for 30 min, occasionally resuspending the beads.
At the final of reaction, wash 5 x with PBS to remove unincorporated ATP.
* Protein Kinase Sigma #P2645, 250 U/vi. It comes lyophilized, resuspend in
25 ul 40 mM DTT in dH2O. Allow it go into solution for 15 min on ice. It
goes bad after a few days at 4C.
4. Generate labeled native Ub. Add 3 x beads volume of 1/20 thrombin stock in
PBS (Pharmacia kit #27-4570-01, diluted into 0.5 ml cold PBS and stored in
small aliquots at –70C). Tumble at RT for 2-3 hr. Spin out beads. Pure
labeled Ub is in supe. Add 100 ul washed benzamidine sepharose beads
(Pharmacia) to supe (this will remove thrombin). Tumble at RT for 30-60 min.
Count 1-2 ul. Expect 20,000-40,000 cpm/ul.
Ubiquitination assay of E3
Binding of MBP-SINAt5 to beads. Add 40 ul prewashed beads to 0.5-1 ml crude
extract and RT rotation for 1 hr for binding. Wash 3 x 1 ml Tris HCl pH7.5 and
remove final wash by very thin tip.
Ubiquitination reaction: Add to the tube the following reagents
20 x buffer*
1.5 ul
E1
3 ul
E2
3 ul
32P-Ub
1 ul
H2O
21.5 to total 30 ul
Incubate at 30C with agitation for 1 hr 30 min. Add 10 ul 4 x protein loading buffer
and heating for protein loading for SDS-PAGE gel.
*20 x buffer
1 M Tris pH 7.5
40 mM ATP (Sigma)
100 mM MgCl2
40 mM DTT
Ubiquitination assay of subtract
Binding of MBP-SINAt5 to beads. Add 40 ul pre-washed beads to 0.5-1 ml crude
extract and RT rotation for 1 hr for binding. Wash 3 x 1 ml Tris-HCl pH 7.5 and
remove final wash by very thin tip. The MBP-SINAt5 on beads work as a E3.
Ubiquitination reaction: Add to the tube the following reagents
20 x buffer*
1.5 ul
E1
3 ul
E2
3 ul
Ub(Sigma)
1 ul
TNT subtract mix
2-8 ul
H2O
to total 30 ul
Incubate at 30C with agitation for 1 hr 30 min. Add 10 ul 4 x protein loading buffer
and heating for protein loading for SDS-PAGE gel.