A. Southwestern blot
Material :
0.22 µm BA83 or BAS83 nitrocellulose membrane (Schleicher & Schuell)
[32P]-NAD = 1 µCi/ml (NEN Dupont de Nemours, ref. NEG-023)
Solutions :
Standard solutions for SDS-PAGE
Loading buffer (for protein reduction before gel loading) :
|
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 Binding buffer (500 ml) :
|
Volume
|
Final
|
| Tris 2 M pH 8.0 |
12.5 ml
|
50 mM
|
| DTT 1 M |
500 µl
|
1 mM
|
| Tween 20 |
1.5 ml
|
0.3%
|
| NaCl 5 M |
15 ml
|
150 mM
|
If required, add 100 ml 100 mM ZnCl2 (20 µM final)
H2O qsp 500 ml
Protocol description :
1. Run your samples on a suitable SDS-polyacrylamide gel (size 11 x 10 cm, i.e.)
2. Blot the proteins onto a nitrocellulose membrane by electrophoretic transfer using 1x Transfer buffer for 1 hr at 200 mA in the cold room
3. Renature the proteins by incubation of the blot in 1x binding buffer for 30 mn at 20°C
4. Incubate the blot in binding buffer containing the [32P]-labelled DNA probe (105 cpm/ml)
5. Wash 3 times for 10 mn at 0°C under agitation
6. Place the blot under autoradiography for 1 hr at 4°C (if followed by footprint). Note : at this step, one can choose to perform either a footprint or a Western)
References :
• Mazen et al. (1989) Poly(ADP-ribose)polymerase: a novel finger protein.
• Ménissier-de Murcia et al. (1989) Zinc-binding domain of poly(ADP-ribose)polymerase participates in the recognition of single strand breaks on DNA.
B. Footprint
Solutions :
Footprint buffer (10 ml)
|
Volume
|
Final
|
| Tris 2 M pH 8.0 |
100 µl
|
20 mM
|
| NaCl 5 M |
140 µl
|
70 mM
|
| CaCl2 50 mM |
20 µl
|
0.5 mM
|
|
DTT 1M
|
5 µl
|
0.5 mM
|
| EDTA 0.2 M |
5 µl
|
0.1 mM
|
qsp H2O 10 ml
Stop buffer (10 ml)
|
Volume
|
Final
|
| Na acetate 3 M |
2 ml
|
0.6 M
|
| SDS 10 % |
2 ml
|
2 %
|
| EDTA 0.2 M |
1ml
|
20 mM
|
|
tRNA
|
50 µl
|
25 ng/µl
|
| EDTA 0.2 M |
5 µl
|
0.1 mM
|
qsp H2O 10 ml
DNase stock solution at 20 mg/ml (Grade II DNase, Boehringer, ref 104159, 100mg)
Phenol/chloroform (v/v)
Protocol description :
1. On the nitrocellulose blot, cut out the radiolabelled band that corresponds to the protein complexed to the DNA probe
2. Incubate this piece of nitrocellulose in 200 ml of footprint buffer for 5 min at 20°C
3. Add 10 µl of a 2 mg/ml Dnase solution ; incubate 2 to 10 min at room temperature
4. Add 200 µl of stop solution ; leave 5 min at 70°C
5. Remove the nitrocellulose and treat the working solution with 200 µl phenol/chloroform ; centrifuge at 10,000 g for 3 min
6. Add 1 ml of cold ethanol to the aqueous phase ; place at –20°C for 30 min ; centrifuge at 10,000 g for 15 min
7. Wash and centrifuge the pellet 3x with 80 % cold ethanol
8. Lyophylize the pellet
9. Count the cpm left ; start the sequencing
Reference :
Huet & Sentenac (1987) : "TUF, the yeast DNA binding factor specific for UASrpg upstream activating sequences: inditification of the protein and its DNA-binding domain"
|
