Sauer:In vitro degradation of GFP-ssrA by ClpXP

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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)

  • Many people leave out the detergent and see similar activity *Jhdavis 12:03, 9 May 2008 (EDT):

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.
    • 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

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

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