Sauer:In vitro degradation of GFP-ssrA by ClpXP: Difference between revisions

Revision as of 11:11, 16 August 2005

25 mM HEPES-KOH, pH 7.6

5 mM MgCl₂

0.032% NP-40 (Nonidet P40 substitute)

10% glycerol

4 mM ATP

16 mM creatine phosphate

0.32 mg/mL creatine kinase

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.
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.

Andreas, Greg, Dan, Kathleen

@@ Line 1: / Line 1: @@
+==Protocol 1==
+([[user:kathmc|Kathleen]], [[user:bolon|Dan]])
+===PD buffer (1X)===
+mM HEPES-KOH, pH 7.6
+mM MgCl<sub>2</sub>
+.032% NP-40 (Nonidet P40 substitute)
+% glycerol
+*Make 4X stock and store at -20 ˚C.
+===ATP regeneration mix (1X)===
+mM ATP
+mM creatine phosphate
+.32 mg/mL creatine kinase
+*Make 10X stock and store at -20 ˚C.
+===Reaction===
+X PD buffer
+mM KCl
+X ATP regeneration mix
+nM ClpX<sub>6</sub>
+nM ClpP<sub>14</sub>
+.15–10 µM GFP-ssrA
+*For GFP-ssrA degradation followed by fluorescence, use 60 µL reaction.
+*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.
 ==Who to ask about this protocol==
 Andreas, Greg, Dan, [[user:kathmc|Kathleen]]
 [[Sauer Lab | Back to the Sauer lab]]