Bio-Rad Criterion™ Blotter - Manual (Page 13)

Section 4
Strategies for Optimizing Electro-Elution
4.1 Optimizing Protein Transfer
Generally, quantitative elution of denatured high molecular weight proteins is difficult. The
following tactics, alone or in combination, will increase transfer efficiency.
1. 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
roller should assure good contact. Proper selection of filter paper spacers will help assure
good compression. Gel and membrane equilibration in transfer buffer for at least 15 minutes
prior to transfer will help prevent shrinking or swelling of either component during
transfer, and will eliminate reactants such as urea or SDS from the gel.
2. Increase transfer time. An initial control should be performed to determine the time
required for complete transfer. 17,24 Times may vary from as little as 30 minutes to as long
as overnight. Remember all overnight applications should be performed at 30–50 volts to
minimize heating problems. (For long transfers at elevated voltages use the Super
Cooling Coil.)
3. Increase the field strength. Initial controls should be performed to evaluate the efficiency
of increasing the V/cm as well as its effects on the temperature of transfer. The
temperature increase may change buffer resistance and subsequent power delivered, as
well as the state of protein denaturation, thus affecting transfer efficiency.
4. Vary buffer type and pH
a. 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.
b. 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.
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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 0.01% to SDS detergent to Tris, glycine, methanol buffer has been reported
to increase transfer efficiency. 24 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 nitrocellulose membranes.
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e. Eliminate alcohol from the transfer buffer. Alcohol in the transfer buffer is required
for binding of SDS proteins to nitrocellulose. Elimination of alcohol results in
increased transfer efficiency but diminishes binding to nitrocellulose. Transfer
efficiency is decreased 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 may reduce the alcohol requirement, and
constitutes a logical strategy for analysis of high molecular weight or
difficult-to-transfer proteins.
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