Sauer:In vitro degradation of GFP-ssrA by ClpXP: Difference between revisions
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0.15–10 µM GFP-ssrA | 0.15–10 µM GFP-ssrA | ||
*For GFP-ssrA degradation followed by fluorescence, use 60 µL reaction. | *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. | *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. | *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. | |||
*Keep everything on ice until you are ready to do the reaction. | |||
===Measuring degradation=== | |||
*Get instructions on how to use the [[Sauer:Fluorimeter|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. | |||
==Who to ask about this protocol== | ==Who to ask about this protocol== |
Revision as of 11:29, 16 August 2005
Protocol 1
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.
- 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.
Who to ask about this protocol
Andreas, Greg, Dan, Kathleen