Preparation of Cell Lysate for Western Blot - What did I do wrong? (Nov/12/2007 )
yep,, starting from now i boil and microcentrifuge my samples before SDS-PAGE.
thanks!
-chrismartin-
low sized polypeptides are missing; presumably blotted through? reduce blotting time
-The Bearer-
QUOTE (The Bearer @ Nov 17 2007, 09:32 PM)
low sized polypeptides are missing; presumably blotted through? reduce blotting time
i normally blot for 1.5 h. i'm not sure that's too long 'cause i can still see some of the prestained protein ladder on the gel.
is there an optimal blotting time?
-chrismartin-
QUOTE (chrismartin @ Nov 20 2007, 05:35 AM)
QUOTE (The Bearer @ Nov 17 2007, 09:32 PM)
low sized polypeptides are missing; presumably blotted through? reduce blotting time
i normally blot for 1.5 h. i'm not sure that's too long 'cause i can still see some of the prestained protein ladder on the gel.
is there an optimal blotting time?
do you use nitrocellulose? it is more critical than PVDF for long transfer time; we use 45 min (~ 500 mA) for semidry blotting
-The Bearer-
I use PVDF membrane (Immobilon brand), semi-dry blotting, 80 mA for 1.5 h.
-chrismartin-