Bio-Rad Trans-Blot® Plus Cell - Manual (Page 24)

4. Vary buffer type and pH.
a. Reduce the 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.
b. b. Maximize the charge-to-mass ratio. Alcohols present in SDS transfer
buffer strip SDS from proteins. Basic proteins in Tris, glycine, and
methanol buffer at pH 8.3 may assume a state near isoelectric neutrality
and thus, may transfer poorly. Buffers with pH of 9.5 to 10.0 have shown
much better elution and binding characteristics for basic proteins such as
lysozyme and histones
3 .
c. Different buffer types at similar V/cm may yield different efficiencies.
Generally Tris buffers allow more efficient transfer than acetate or
phosphate buffers.
d. Addition of 0.1% SDS detergent to Tris, glycine, and methanol buffer has
been reported to increase transfer efficiency
2 . SDS, however, increases
relative current, power, and heating. 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
4 .
e. Alcohol in the transfer buffer has opposing effects on the efficacy of trans-
fer. Alcohol in the transfer buffer removes SDS from protein-SDS
complexes and increases the affinity between proteins and nitrocellulose
membranes. Alcohol also causes a reduction in gel pore size, restricting
transfer of some proteins. Alcohol may also cause some proteins to
precipitate and transfer inefficiently. Proteins bind efficiently to PVDF
membrane in the absence of alcohol. Therefore, elimination of alcohol
from the transfer buffer and use of PVDF membrane for SDS-protein
transfers may constitute a logical strategy for analysis of high molecular
weight or difficult-to-transfer proteins
5,6 . Alcohol is not required in the
transfer buffer when proteins are being transferred from gels not
containing SDS.
5. Alter membrane type.
As mentioned in 4e, PVDF membrane allows transfer in the absence of
alcohol. PVDF can increase the binding of low molecular weight proteins that
sometimes blow through nitrocellulose when transfers are long enough or
intense enough to transfer high molecular weight proteins. Use Immun-Blot
PVDF if the blot will be developed with immunochemicals. Use Sequi-Blot
PVDF is the proteins will be sequenced or analyzed by mass spectrometry.
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