Complete transfer, can it be done or not? - (May/07/2009 )

Hi all,

I am using for quite some time now the semidry system from BIORAD to transfer my gels. At some point I had some trouble with the signal but now is everything ok.
Because i was not so sure whether the system was working ok or not I called up the BIORAD and they told me that there is no such thing as complete transfer. Basically they said that all is very relative depending on the AS sequence and load of my protein and bla bla bla.
Does anybody have any experience with semidry transfer or is using the same machine? Is the term complete transfer totally off or is i sth that should always happen? Any experienced proteomics Prof out there?

thank you very much

If you want a more complete transfer, you need to go wet.

Transfer does depend on the AA sequence, the molecular weight and loading, as well as buffer components, how much voltage/amperage you are using, presence of fixing agents, etc. What works best will have to be determined empirically for your system.

bob1 on May 7 2009, 04:44 PM said:

Slower, colder, and more even distribution of current - better conditions for the proteins too.

I agree with Bob, I get great transfer using the following, esp for high molecular weight proteins:

3g Tris base
14.9g glycine
NO METHANOL

equilibrate gel in transfer buffer (above) for ~30min

transfer (wet) onto methanol-activated PVDF (rinsed in diH2O, then in transfer buffer) for 15V, o/n at 4C

Sounds like a really long transfer and it is, but this works well esp. if you are trying to get higher MW bands. Good luck!