Material :
0.22 µm BA83 or BAS83 nitrocellulose membrane (Schleicher & Schuell)
[32P]-NAD = 10 µCi/µl (Amersham)
Activated DNA (DNase I - treated DNA)
Solutions :
Standard solutions for SDS-PAGE
Reduction buffer (for protein reduction before transfer), 300 ml:
|
Volume
|
Final
|
| 6x transfer buffer (see below) |
50 ml
|
1x
|
| ß mercapto-ethanol 14 M |
15 ml
|
0.7M
|
| H2O |
235 ml
|
|
Aliquote and store at –20°C
6x Transfer buffer (2 liters)
|
weight
|
conc.(6x)
|
conc.(1x)
|
| Tris base |
36.33 g
|
150 mM
|
25 mM
|
| glycine |
173 g
|
1152 mM
|
192 mM
|
| SDS |
12 g
|
0.6 %
|
0.1 %
|
qsp H2O 2 litres pH 8,3
1x Renaturation buffer (20 ml) :
|
Volume
|
Final
|
| Tris 2 M ph 8.0 |
0.5 ml
|
50 mM
|
| DTT 1 M |
20 µl
|
1 mM
|
| Tween 20 |
60 µl
|
0.3%
|
| NaCl 5 M |
0.6 ml
|
150 mM
|
| Zn acetate 100mM |
4 µl
|
20 µM
|
| MgCl2 M |
40 µl
|
2 mM
|
Activated DNA : 2µg/ml (final concentration)
H2O qsp 20 ml
Protocol description :
1. Run your samples on a suitable SDS-polyacrylamide gel (size 11 x 10 cm, i.e.)
2. Incubate the SDS-polyacrylamide gel for 1 hr at 37°C under agitation in reduction buffer
3. Blot the proteins onto a nitrocellulose membrane by electrophoretic transfer using 1x Transfer buffer for 1 hr at 200 mA in the cold room
4. Renature the proteins by incubation of the blot in 1x renaturation buffer for 1 hr at 20°C under agitation
5. Incubate the blot in 1x renaturation buffer in the presence of [32P]-NAD for 1 hr at 20°C under agitation
6. Wash 2x for 30 min at 20°C in 1x renaturation buffer
7. Place the blot under autoradiography
References :
Simonin et al. (1990) Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity.
Simonin et al. (1991) Detection of poly(ADP ribose) polymerase in crude extracts by activity-blot.
|
