Bio-Rad Mini Trans-Blot® Cell - Manual (Page 22)

16 Mini-Trans-Blot Electrophoretic Transfer Cell
Reduce buffer strength
Dilution of transfer buffer results in lower current at any
given voltage. This will allow the use of higher voltages
without excessive heating. However, be aware not to dilute
the buffer below its buffering capacity.
Vary buffer type and pH
Maximize charge-to-mass ratio. It appears that alcohols
present in SDS transfer buffer strip SDS from proteins.
Basic proteins in Tris, glycine, methanol buffer at pH
8.3 may assume a state near isoelectric neutrality and
thus transfer poorly. For example, lysozyme exhibits this
behavior. Buffers with pH of 9.5–10.0 have shown much
better elution and binding characteristics for basic proteins
such as lysozyme and histones.
41
Different buffer types at similar V/cm may yield different
efficiencies. Generally, Tris buffers allow more efficient
transfer than acetate or phosphate buffers.
Add detergent
Addition of 0.1% SDS detergent to Tris, glycine, methanol
buffer has been reported to increase transfer efficiency.
25
SDS, however, increases relative current, power, and
heating. Also, temperatures below 10°C may precipitate
the SDS so the starting buffer temperature will be higher.
SDS may also affect the antigenicity of some proteins.
SDS will aid in eluting the proteins from the gel, but it
may reduce the binding efficiency of those proteins to the
membrane.
Eliminate alcohol from the transfer buffer
Alcohol in the transfer buffer improves binding of proteins
to nitrocellulose only. Elimination of alcohol results in
increased transfer efficiency but diminishes binding to
nitrocellulose. Transfer efficiency is increased because
alcohol causes gel pores to contract resulting in capture of
large molecular weight proteins within the gel matrix.