Proteasome overlay

1. Prepare native PAGE minigel:

40% acrylamide-bisacrylamide (37.5:1) 1 ml
1 M Tris-borate pH 8.3 1.8 ml
1 M MgCl2 50 ml
100 mM ATP 0.1 ml
1 M DTT 10 ml
ddH2O 7.0 ml
10% ammonium persulfate 0.1 ml
TEMED 10 ml
Total volume 10 ml

2. Add xylene cyanol FF (0.01%) to the proteasome samples or run in a separate lane.

3. Load proteasome sample and run using the running buffer (0.18 M Tris-borate pH8.3, 5 mM MgCl2, 1 mM ATP, 1 mMDTT).

4. Stop the gel when the xylene cyanol dye reaches the end.

5. Transfer the gel to a dish and overlay with 10 ml of 0.1 mM Suc-LLVY-AMC in 50 mM Tris-HCl pH7.4, 5 mM MgCl2, 1 mM ATP, 1 mMDTT, 10% glycerol.

6. Incubate at room temperature or 37 °C for 10-30 minutes.

7. Visulize under a UV light.