3 replies to this topic

[bio01418]

Hi,everyone. Does anyone has a detail protocol for membrane transfer after native-page? Thanks.

[mdfenko]

just follow the same protocol that you use for sds-page unless you are separating basic proteins.

also, methanol is not required in the transfer buffer unless you add sds to facilitate transfer.

methanol is still required to activate a pvdf membrane (not for nitrocellulose).

[bio01418]

mdfenko, on Jan 8 2010, 08:30 AM, said:

just follow the same protocol that you use for sds-page unless you are separating basic proteins.

also, methanol is not required in the transfer buffer unless you add sds to facilitate transfer.

methanol is still required to activate a pvdf membrane (not for nitrocellulose).

Thanks，mdfenko. And I do want to separate a basic protein form cell extract, of which the PI is 9.0. Do you have a protocol for this situation?

[mdfenko]

you can use the same buffer but put the membrane on the other side

or

you can try a caps buffer (normally used at pH 11, adjusted with naoh, and 10mM working concentration).