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Electrophoretic transfer is by far the most widely used transfer method. This method uses an electrical current to transfer proteins to a membrane for subsequent protein analysis. The transfer buffer, on the other hand, provides electrical continuity between the electrodes and must be conductive. It also provides a chemical environment that maintains protein solubility without hindering protein adsorption to the membrane during transfer.
Figure 1. Buffer in the wet-blot experimental apparatus.
(a) Towbin system buffer (25 mM Tris, 192 mM glycine, 20% methanol (v/v), 0-0.01% SDS).
The glycine used in the Towbin buffer results in high background in automated protein sequencers using the Edman principle. Therefore, this transfer buffer system cannot be used in experimental projects involving protein sequencing.
(b) CAPS buffer system (CAPS: 10 mM 3-(cyclohexyl-amino)-1-propanesulfonic acid, 10% methanol (v/v), pH 11).
CAPS buffer is suitable for western blot of high molecular weight proteins (greater than 20KD). Since the electrophoresis buffer made of CAPS only contains CAPS and methanol, the PVDF membrane of protein gel electrophoresis is not only used for protein separation, but also for subsequent protein sequencing.
In general, transfer buffers without SDS work better when using PVDF membranes, as proteins can cross the membrane planes in the presence of SDS, causing inaccurate results. However, for proteins that tend to precipitate, it is best to add SDS (<0.01 %) to the buffer during the transfer and fine-tune the transfer time, current, etc. to get a clear and accurate result.
Polyacrylamide is a hydrogel with the ability to absorb water. In pure water, there is a considerable increase in gel size in all dimensions. The degree of swelling also depends on the concentration of acrylamide used in the gel. High concentration gels swell more than low concentration gels. Gradient gels will accentuate this effect significantly, with areas of high concentration at the bottom swelling more than the top. Gels that start out as rectangular may become trapezoidal. Methanol added to the transfer buffer minimizes gel swelling, and transfer operations often also include an equilibration step for gel dimensional stability. Dimensional stability is achieved fairly quickly at methanol concentrations of 10%-20%. Equilibrium takes longer to achieve when the methanol concentration is lower. Protein resolution may be lost if size changes occur during transfer. For high-molecular-weight proteins with limited solubility in methanol, the reduction of methanol results in significantly higher protein transfer efficiency, but this may require longer equilibration times to ensure dimensional stability.
A second function of methanol is critical for the transfer of proteins from gels containing SDS. Methanol helps dissociate complexed SDS from protein molecules. Although SDS is necessary for the resolution of individual proteins on a gel, it is extremely detrimental for efficient blotting. First, the binding of SDS brings a high negative charge density to the protein molecule, which causes the protein molecule to pass through the membrane very quickly, reducing the stay in the pore structure, thereby reducing the chance of intermolecular interaction. Second, by coating protein molecules, SDS limits the ability of proteins to make molecular contacts with PVDF. These effects increase as the molecular weight of the protein decreases. Methanol reduces both effects by dissociating SDS and increasing the likelihood of protein molecules binding to the membrane.
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