Sauer:In vitro degradation of GFP-ssrA by ClpXP: Difference between revisions
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== | {{Sauer lab sidebar}} | ||
==Protocol 1== | |||
===PD buffer (1X)=== | |||
[[ | 25 mM HEPES-KOH, pH 7.6 | ||
5 mM MgCl<sub>2</sub> | |||
0.032% NP-40 (Nonidet P40 substitute) | |||
* Many people leave out the detergent and see similar activity *'''[[User:Jhdavis|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) - [[user:jhdavis|Joey]] | |||
===Reaction=== | |||
1X PD buffer | |||
200 mM KCl | |||
1X ATP regeneration mix | |||
100 nM ClpX<sub>6</sub> | |||
300 nM ClpP<sub>14</sub> | |||
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.--[[user:kathmc|Kathleen]] | |||
*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. | |||
'''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 |
Latest revision as of 10:53, 13 March 2009
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