hello,

Try reducing the time of transfer to approximately 10-15min at 100mA. Reason, protein is fairly small, so it will traverse the membrane if left to transfer for an extended period of time. best of luck .

Joshua

hi
I had this problem before, then I used a PVDF (0.45umpore size)
I activate the membrane for 2minutes in 100% methanol, then soak the membrane in transfer buffer for 5-10 minutes, I transfer at 20Volts for 20 minutes, and I always get a good result. But wet transfer is the best specially for 2-D gels.

regards

so you basically want to say that you are having problem with the semi dry western condition...i dont think that this is only the cause...is there any other thing which you are hesitant to say...

flashboy on Aug 31 2004, 05:32 PM said:

hey flashboy, when using your semi-dry system, did you ever get transfer of anything (i.e. makers?) Is anyone in the lab getting successful transfer with the semi-dry blotter but you? If the answer to both this questions is no, my bet is your blotter is faulty, and you've been banging your head for the wrong reasons. Happened to me in the past, very very annoyed to waste my time "optimising". Also, make sure the transfer buffer is fresh, as changes in pH, salt content, and MeOH evaporation will make it not work.

I can perfectly understand why you dont want to ever use semi-dry again after this, but as I've always got better results with semi-dry I thought it was worth telling you my experience

Had the same problems with a dry blotter once, the iBlot from Invitrogen. Couldn't get a decent transfer of a phospho-protein no matter how hard I tried and then I tried wet once and it worked perfectly. Haven't touched the iBlot again since that.