Sauer:In vitro peptide degradation by ClpXP
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Protocol 1
For peptides containing Abz and Y(NO2)
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, but it helps prevent protein sticking to tubes *
10% glycerol
- 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.
Reaction
1X PD buffer
200 mM KCl
1X ATP regeneration mix
800 nM ClpX6
300 nM ClpP14
0.5 - 120 µM GFP-ssrA
- For peptide 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 peptide you are using.
- Be sure to equalize the salts that may come in with your proteins/peptides (SspB, etc.) across reactions because ClpX is very salt sensitive.
- Make a mix of everything except peptide and SspB such that you will add an equal amount of the master mix to each reaction. I usually make my Substrate+SspB equal to 20 µL and add 40 µL of the mix to each reaction.--
- Keep everything on ice until you are ready to do the reaction.
Measuring degradation
- Get instructions on how to use the fluorimeter.
- excitation wavelength = 320 nm, emission wavelength = 420 nm
- water bath at 30 ˚C
Pre-warm cuvette in cuvette holder for at least 5 min prior to each reaction.
Pre-warm peptide (and SspB, if using) in water bath for 2 min.
Pre-warm ClpX mix for reaction in a tube in the water bath or in cuvette.
Add substrate to ClpX in cuvette and mix well, trying not to introduce any bubbles.
Start measuring fluorescence and measure for ~3 min to get initial rate (beware of bubbles of equilibration artifacts).
Slit width
- Set all four the same, it's easier that way.
- I recommend ~ 3.5 half turns of the screw.
