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SAUER LAB

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Contents

Protocol 1

PD buffer (1X)

25 mM HEPES-KOH, pH 7.6

5 mM MgCl2

0.032% NP-40 (Nonidet P40 substitute)

10% glycerol

ATP regeneration mix (1X)

4 mM ATP - must be pH to 7.0

16 mM creatine phosphate

0.32 mg/mL creatine kinase

Reaction

1X PD buffer

200 mM KCl

1X ATP regeneration mix

100 nM ClpX6

300 nM ClpP14

0.15–10 µM GFP-ssrA

Measuring degradation

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

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