Bio-Rad Trans-Blot® Cell - Manual (Page 16)

5. Vary buffer type and pH
a. 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 to 10.0 have shown much better elution
and binding characteristics for basic proteins such as lysozyme and histones.
41
b. Different buffer types at similar V/cm may yield different efficiencies. Generally
Tris buffers allow more efficient transfer than acetate or phosphate buffers.
6. Addition of 0.1% SDS detergent to Tris, glycine, methanol buffer has been reported to
increase transfer efficiency. 24 SDS, however, increases relative current, power, and heat-
ing. Also, temperatures below 10 °C may precipitate the SDS so the starting buffer tem-
perature 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 nitrocellulose membrane.
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7. Eliminate alcohol from the transfer buffer. Alcohol in the transfer buffer improves bind-
ing of SDS 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 fixation of large molecular weight
proteins within the gel matrix. Use of PVDF membrane for SDS protein transfers elimi-
nates the alcohol requirement, and constitutes a logical strategy for analysis of high molec-
ular weight or difficult-to-transfer proteins.
26, 27
8. Limited protease treatment. A protocol for protease digestion of protein during transfer has
been published. 22 Efficient transfer without loss of immunological reactivity was reported.
9. Alter membrane type. As mentioned in 7, PVDF membrane allows transfer in the absence
of alcohol.
10. Alter gel system. If possible, use non-denaturing gradient pore gels for separation of pro-
teins by molecular weight. Isoelectric focusing gels, or native gels, may be considered if
separation by molecular weight is not mandatory.
11. 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 30 minutes to 1 hour prior to transfer will help prevent shrinking of either com-
ponent during transfer, and will eliminate reactants such as urea or SDS from the gel.
4.2 Optimizing DNA and RNA Transfer
Problems with elution of nucleic acids can be solved by altering the gel percentage. It
may be somewhat more difficult to quantitatively transfer large amounts of DNA used in
genomic blots. The following tactics should be considered for optimizing elution in such
transfers.
1. Alter gel composition.
a. Lower % total monomer or % crosslinker for polyacrylamide gels.
b. Lower % agarose. This allows better elution of high molecular weight DNA.
2. Alter DNA denaturants. It has been found that glyoxal denaturation allows more efficient
elution of DNA than NaOH. Boiling polyacrylamide gels to denature DNA has also been
found to give excellent results.
11 Base denaturation often causes polyacrylamide gels to
weaken and stick to blotting membranes.
13