Material :
0.22 µm BA83 or BAS83 nitrocellulose membrane (Schleicher & Schuell)
65ZnII (NEN Dupont de Nemours ref. NEZ109, 500 µCi)
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 Transfert buffer (600 ml) :
|
Volume
|
Final
|
| 6x transfer buffer (see above) |
100 ml
|
25 mM
|
| ethanol |
120 ml
|
20 %
|
| H2O |
600 ml
|
|
Renaturation buffer (100 ml) :
50 mM Tris-HCl buffer at pH 7.5
Zinc incubation buffer (100 ml) :
|
Volume
|
Final
|
| Tris-HCl 1 M pH 7.5 |
5.5 ml
|
50 mM
|
| KCl 1 M |
10 ml
|
100 mM
|
| H2O |
85 ml
|
|
Protocol description :
1. Run your samples on a suitable SDS-polyacrylamide gel (size 11 x 10 cm, i.e.)
2. Reduce the proteins following migration by incubation of the gel in reducing buffer for 1 hr at 37°C
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 soaking the blot in renaturation buffer for 1 hr at 4°C
5. Wash the membrane for 15 min in zinc incubation buffer at 4°C
6. Incubate the blot in 5 ml of zinc incubation buffer containing 1 mCi of 65ZnII for 15 min at 20°C
7. Wash the membrane 3 times for 10 min in zinc incubation buffer at 20°C under agitation
8. Place under autoradiography for 1 to 24 hr
Reference :
Mazen et al. (1988) Anal. Biochem. 172, 39-42.
|
