*化01群小川型霍亂弧菌抗體IgG

Assemble "sandwich" for Bio-Rad's Transblot. 
Prewet the sponges, filter papers （slightly bigger than gel） in 1x Blotting buffer.
Sponge - filter paper - gel - membrane - filter paper - sponge 
Transfer for 1 hr at 1 amp at 4°C on a stir plate. 
Bigger proteins might take longer to transfer. 
For the Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer. 
When finished, immerse membrane in Blocking buffer and block overnight. 

H. Antibodies and detection 

Incubate with primary antibody diluted in Blocking buffer for 60 min at room temp. 
Wash 3 x 10 min with 0.05% Tween 20 in PBS. 
Incubate with secondary antibody diluted in Blocking buffer for 45 min at room temp. 
Wash 3 x 10 min with 0.05% Tween 20 in PBS. 
Detect with Amersham ECL kit （RPN 2106）. 

I. Stripping blot 

Rinse blot off with 0.05% Tween 20 in PBS. 
Put blot into Kapak bag cut to slightly bigger size than blot. 
Add about 5 to 10 ml Stripping buffer. 
Remove as much air as possible and seal bag. 
Immerse into 80°C water bath and incubate for 20 min. 
Rinse blot off with 0.05% Tween 20 in PBS. 
Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.