Sauer:In vitro degradation of GFP-ssrA by ClpXP
PD buffer (1X)
25 mM HEPES-KOH, pH 7.6
5 mM MgCl2
0.032% NP-40 (Nonidet P40 substitute)
- Many people leave out the detergent and see similar activity *Jhdavis 12:03, 9 May 2008 (EDT):
- Make 4X stock and store at -20 ˚C.
ATP regeneration mix (1X)
4 mM ATP - must be pH to 7.0
16 mM creatine phosphate
0.32 mg/mL creatine kinase
- Make 10X stock and store at -20 ˚C.
- I make up a 200 mM ATP stock pH to 7.0, a 640 mM creatine phosphate stock and a 32 mg/mL creatine kinase stock. These are then added appropriately to a 2x degradation buffer stock to give 10x atp regen mix in 1x degradation buffer (0.2 mL atp/mL final, 0.25 mL CP/ml final, 0.05 mL CK/mL final, 0.5 mL 2x buffer/ml final) - Joey
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, 0.3 mm cuvette.
- 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.
- Make a mix of everything except GFP and SspB such that you will add an equal amount of the master mix to each reaction. I usually make my GFP+SspB equal to 20 µL and add 40 µL of the mix to each reaction.--Kathleen
- Keep everything on ice until you are ready to do the reaction.
- Get instructions on how to use the fluorimeter.
- excitation wavelength = 467 nm, emission wavelength = 511 nm
- water bath at 30 ˚C
Pre-warm cuvette in cuvette holder for at least 5 min prior to each reaction.
Pre-warm GFP (and SspB, if using) in cuvette for 2 min.
Pre-warm ClpX mix for reaction in a tube in the water bath.
Add warmed ClpX mix to GFP in cuvette and mix well, trying not to introduce any bubbles.
Start measuring fluorescence and measure for ~10 min, or until reaction is finished.
- Set all four the same, it's easier that way. 1 turn = 1 full turn of the screw.
- This is just a guideline. Test it out to see what works best for your samples.
5 µM GFP 0.75 turn
2.5 µM GFP 1 turn
1.2 µM GFP 1.25 turns
0.6 µM GFP 1.5 turns
0.3 µM GFP 1.75 turns
0.15 µM GFP 2 turns