Use of a discontinuous buffer system, that utilizes different buffer formulations on the anode and the cathode side of the transfer sandwich (see Transfer Buffers) may enhance semi-dry transfer of high molecular weight proteins (>80 kD). Moreover, because low buffer capacity limits run times, some large proteins may be poorly transferred. Under semi-dry conditions, some small proteins may be driven through the membrane in response to the high field strengths. As a result, high electric field strengths and high-intensity blotting conditions are achieved. In semi-dry systems, the distance between the electrodes is limited only by the thickness of the gel and membrane sandwich. The term semi-dry refers to the limited amount of buffer, which is confined to the two stacks of filter paper. In a semi-dry transfer, the gel and membrane are sandwiched between two stacks of filter paper that are in direct contact with plate electrodes (Bjerrum and Schafer-Nielsen 1986, Kyhse-Andersen 1984, Tovey and Baldo 1987). Active cooling options are limited with semi-dry blotting Semi-dry systems - gels and membranes are sandwiched between buffer-saturated filter papers that are in direct contact with plate electrodes these systems are typically easier to set up than tank systems and are useful when high-throughput is necessary and extended transfer times are not required, or when discontinuous buffer systems are used.Tank transfer systems offer the most flexibility in choosing voltage settings, blotting times, and cooling options Tank transfer systems - gels and membranes are submerged in transfer buffer in tanks these systems are useful for most routine protein work, for efficient and quantitative protein transfers, and for transfers of proteins of all sizes.There are two main types of electrophoretic blotting apparatus and transfer procedures (see table below): * Proteins denatured with sodium dodecyl sulfate (SDS) carry a net negative charge and migrate toward the anode. The major limitation of any electrophoretic transfer method is the ability of the chamber to dissipate heat. In addition, excessive heat may cause the gel to deteriorate and stick to the membrane. Such changes in resistance may lead to inconsistent field strength and transfer, or may cause the transfer buffer to lose its buffering capacity. Joule heating increases temperature and decreases resistance of the transfer buffer. The heat generated (Joule heating) is proportional to the power consumed by the electrical elements (P), which is equal to the product of the current (I) and voltage (V): There are practical limits on field strength, however, due to the production of heat during transfer. A number of other factors, including the size, shape, and charge of the protein*, the pH, viscosity, and ionic strength of the transfer buffer, and the composition of the gel, also influence the elution of particular proteins from gels. Both the applied voltage and the distance between the electrodes then play a major role in governing the rate of elution of the proteins form the gel. The electric field strength (E, measured in V/cm) that is generated between the electrodes is the driving force for transfer. R is the resistance generated by the materials placed between the electrodes (that is, the transfer buffer, gel, membrane, and filter papers). Proteins migrate to the membrane following a current (I) that is generated by applying a voltage (V) across the electrodes* following Ohm's law: Gel and membrane setup for electrophoretic transfer. In an electrophoretic transfer, the membrane and protein-containing gel are placed together, with filter paper between two electrodes. Electrophoretic transfer is the most widely used blotting method because of its speed and precision in replicating the pattern of separated proteins from a gel to a membrane. In electrophoretic transfer, an electric field is used to elute proteins from gels and transfer them to membranes.
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