western of a big protein - (May/24/2005 )
How long do you blot if the protein is 300 kDa? Is 3 hours at 400 mAmp enough?
For large proteins I usually transfer overnight at 35 V (~70mamps) and then crank it up to 100V (~300 mAmps) the following morning for 1 hour. In addition, in my experience (is it just me?) I find I get more efficient transfers if only one sandwich is used per transfer apparatus.
Thanks! I will try your method. yesterday i transferred for 3 H 15m at 400 mAmp but without success. Is 5 % gel ok?