Sauer:In vitro degradation of GFP-ssrA by ClpXP

Protocol 1

(Kathleen, Dan)

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, 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.

Measuring degradation

• 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.

Slit width

• 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

Who to ask about this protocol

Andreas, Greg, Dan, Kathleen

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