Sauer:In vitro degradation of GFP-ssrA by ClpAP
ClpA Activity Buffer (4X)
200 mM HEPES-KOH, pH 7.5
80 mM MgCl2
2mM DTT (add at last minute)
- 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.
1X ClpA Activity buffer
1X ATP regeneration mix
250 nM ClpA6
750 nM ClpP14
0.15–10 µM GFP-ssrA
- For GFP-ssrA degradation followed by fluorescence, use 60 µL reaction, 0.3 mm cuvette.
- Make a mix of everything except GFP asuch that you will add an equal amount of the master mix to each reaction.
- 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 in cuvette for 2 min.
Pre-warm ClpA mix for reaction in a tube in the water bath.
Add warmed ClpA 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