Far Westerns

Far Western Blot (Roberts and Green, 1994, Nature 371, 717; Reese and Green, 1994, Nature 371, 523)

- run SDS-PAGE, transfer onto Nitrocellulose or PVDF membrane conditions: 1 hr at 0.6 A with cooling!; 4 hrs at 150 mA; overnight at 45 mA

- check transfer with PonceauS staining

- wash membrane three times for 5 min in PBS/0.1 % Tween 20 once in PBS

- incubate overnight at 4°C in

* 20 mM Hepes pH 7.5
* 50 mM KCl
* 5 mM MgCl2
* 1 mM EDTA
* 1 mM DTT
* 0.05% NP40
* 17% glycerol
* 5% BSA or 5% dry milk

- probe blot with 10 ml S-35 TNT in same buffer with 0.5 % BSA or 5 % dry milk for 6 hrs at 4°C in K-PAK pouch on nutator before using TNT pass over Sephadex G-25 column

- wash blot twice for 15 min with ice-cold binding buffer on shaker

- dry membrane, wrap in Saran wrap, expose