Sauer:In vitro degradation of GFP-ssrA by ClpXP
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Protocol 1
PD buffer (1X)
25 mM HEPES-KOH, pH 7.6
5 mM MgCl2
0.032% NP-40 (Nonidet P40 substitute)
10% glycerol
- Make 4X stock and store at -20 ˚C.
ATP regeneration mix (1X)
4 mM ATP
16 mM creatine phosphate
0.32 mg/mL creatine kinase
- Make 10X stock and store at -20 ˚C.
Reaction
1X PD buffer
200 mM KCl
1X ATP regeneration mix
100 nM ClpX6
300 nM ClpP14
0.15–10 µM GFP-ssrA
- For GFP-ssrA degradation followed by fluorescence, use 60 µL reaction.
- If you are using SspB, include it at a concentration that is equal to that of the GFP-ssrA you are using.
- Be sure to equalize the salts that may come in with your proteins (GFP, SspB, etc.) across reactions because ClpX is very salt sensitive.
Who to ask about this protocol
Andreas, Greg, Dan, Kathleen