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Preparation
of Pyrenyl-Actin
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In
many experimental situations it is desirable to be able to measure the
extent of actin polymerisation. The pyrenyl fluorescence assay (Kouyama.
& Mihashi, 1981)
is a convenient, non-destructive method for this that is very extensively
used for this. Actin (from
most species) has a reactive cysteine residue at the penultimate position
374, which can be labelled with pyreneiodoacetamide at high pH.
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N-(1-pyrene)iodoacetamide - Available from Molecular
Probes - (Cat
No P-29)
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The labelled G-actin is weakly fluorescent,
but upon polymerisation the fluorescence is enhanced 20 times.
The extent of polymerisation can therefore be followed by measuring
the change in fluorescence using suitable wavelengths in a
spectrofluorimeter. Typically,
the actin being monitored is “doped” with a small amount of the
labelled actin both to conserve this material and to avoid possible
artefacts.
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Method.
1).
Dialyze 30-80 mgs of actin against:-
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Stock |
/litre |
| 25 mM Tris pH 8.0 |
1 M |
25 mls |
| 0.1 M KCl |
- |
7.46
g |
| 2 mM MgCl2 |
1 M |
2 mls |
| 3 mM NaN3 |
1 M |
3 mls |
| 0.3
mM ATP |
0.1 M |
3 mls |
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2).
Clarify a 1ml sample and read the OD290.
Dilute the remainder to 1 mg/ml (= 23.8 µM; or 0.63 OD290);
calculate the total nmoles.
3).
Transfer to a 100ml flask with a magnetic flee.
4).
Make up a 10mM (MW = 385) stock solution in Dimethylformamide (DMF).
Add 4 to 7 mol pyrenyliodoacetamide (Molecular Probes) per mole of actin,
while gently stirring.
5).
Cover with foil and leave overnight at 4oC.
6).
Centrifuge at low speed (JA20, 5000 rpm, 5 min) to pellet the
precipitated dye.
7).
Transfer the cleared sup. to a 42.1 (Beckman) tube and spin at
38000 rpm for 2hrs at 4oC.
8). Homogenise the pellet in
Buffer G to a concentration of about 6 mg/ml.
Briefly sonicate if you have one around (it speeds up
depolymerisation). Dialyze
against buffer G for 2 days, with 2 changes of buffer.
Buffer
G:-
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Stock
|
per 100mls |
100x |
| 2 mM Tris pH
8.0 |
1M |
2
ml
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20ml
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| 0.2 mM ATP |
0.1
M
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2
ml
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20ml
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| 0.5 mM DTT |
dry |
77
mg
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0.77g
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| 0.2 mM CaCl2 |
0.1
M
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2
ml
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20mls |
| 0.002% NaN3 |
dry |
0.2
g
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2g
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9).
Centrifuge at 38000 rpm for 2 hrs at 4oC.
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10).
Gel filter with S-300 in buffer G.
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11).
Read the OD at 344 and 290 for each fraction.
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12).
Calculate the concentrations:-
[pyrene, µM] = OD344 / 2.2 x 104 (M-1)
[Actin, µM] = (OD290 - (OD344 * 0.127)) /
2.66 x 104 (M-1)
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Reference:
Kouyama, T. & Mihashi, K. (1981). “Fluorimetry study of
N-(1-Pyrenyl)iodacetamide-labelled F-actin.” Eur.J.Biochem. 114,
33-38
Cooper,
J.A. and T.D. Pollard, Methods to measure actin polymerization.
Methods Enzymol., 1982. 85, p. 182-210.
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