Materials:
Labelled actin, ATP, Polymerisation buffer are stored in aliquots at -80¡C. Please refer to the
instruction manual by Cytoskeleton for details and troubleshooting.
1. Preparation of the recombinant proteins
Dia proteins, e. g. ZZ-dia-H6 are purified by Ni-affinity chromatography and stored in aliquots in 50
mM Tris/HCl pH8, 50 mM NaCl, 10% glycerol, 250 mM saccharose at -80C. The concentration should be higher
than 1 mg/ml.
2. Depolymerisation of actin
- add 2 ul 100 mM ATP and 5 ul 100 mM DTT to 1000 ul A buffer (yields G buffer)
- add 225 ul of G buffer to 5 ul of an pyrene-actin (500 uM aliquots stored at -80C)
- incubate for 1 h on ice
- spin for 30 min, 4C, 70k (TLA100.3 rotor), use the special (UZ) reaction tubes
3. Protein in A buffer
- the protein has to be transfered to a buffer that does not interfere with the
polymeristation reaction (no KCl, no Mg2+, no ATP, low buffer concentration)
- Dia-FH1FH2 shows activity at concentrations higher than 20 nM.
4. Polymerisation
- the final volume of the reaction mix is 100 ul
- mix 10 ul of polymerisation starter with 60 ul of A buffer with the protein
and load into the cuvette (3 way for fluorometer)
- start the reaction by adding (and mixing) 30 ul of G-actin in G buffer
(final actin concentration is 3 uM)
- excitation is at 365 nm, fluorescense at 407 nm, read every 12,5 s over 500 s.
Test of actin
Frozen globular actin looses its polymerisation ability. To test the quality of the
preparation perform following procedure:
- Dissolve actin to 0.2 mg/ml in water. Incubate for 1h at room temperature.
- Fill the solution into a fluorometer cuvette and read the fluorescence every minure
for 10 min.
- Add 1/10 of 10xpolymerisation buffer. Mix.
- Read the fluorescence every minute for 30 min.
By incubation at room temperature, actin oligomers are formed. After increasing
the Mg concentration and adding K+ ions, their elongation is promoted.
Buffer:
A buffer (1x)
5 mM Tris/HCl pH8
0.2 mM CaCl2
G buffer (1x)
5 mM Tris/HCl pH8
0.2 mM CaCl2
0.2 mM ATP
0.5 mM DTT
ATP stock solution
100 mM, stored at -80¡C
DTT stock solution
100 mM or 1 M, stored at -20¡C
Polymerisation starter (10x)
100 mM Tris/HCl pH 7.5
500 mM KCl
20 mM MgCl2
10 mM ATP
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