Bio-Rad Mini Trans-Blot® Cell - Manual (Page 23)

Mini-Trans-Blot Electrophoretic Transfer Cell 17
Use of PVDF membrane for protein transfers eliminates
the alcohol requirement, and constitutes a logical
strategy for analysis of high molecular weight or difficult-
to-transfer proteins.
27, 28 PVDF must be wetted in 100%
methanol but may then be used in buffer without
methanol.
Limited protease treatment
A protocol for protease digestion of protein during transfer
has been published.
23 Efficient transfer without loss of
immunological reactivity was reported.
Alter membrane type
Both nitrocellulose and PVDF can be used for protein
transfer.
Alter gel system
If possible, use nondenaturing gradient pore gels for
separation of proteins. Isoelectric focusing gels, or native
gels, may be considered if separation by molecular weight
is not mandatory.
Enhance gel-membrane contact
Failure of molecules to bind efficiently to the membrane,
caused by poor gel-membrane contact, is often confused
with inefficient elution. Poor contact is usually due to
excess moisture in the gel-membrane interface. Proper
technique and the use of a test tube or glass pipet as a
“rolling pin” should assure good contact. Proper selection
of filter paper spacers will help assure good compression.
Gel and membrane equilibration in transfer buffer for 15–
20 min prior to transfer will help prevent shrinking of either
component during transfer, and will eliminate reactants
such as urea or SDS from the gel.