Sauer:In vitro degradation of GFP-ssrA by ClpAP

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Contents

Protocol 1

ClpA Activity Buffer (4X)

200 mM HEPES-KOH, pH 7.5

80 mM MgCl2

1.2M NaCl

10% glycerol

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.

Reaction

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.

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 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.

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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