This section describes the theory and some useful equations pertaining to electrophoretic transfer of proteins and provides tips for adjusting power conditions suitable for various types of protein transfers.
Related Topics: Transfer Buffers, Transfer Conditions, Membranes and Blotting Papers, and Protein Blotting Methods.
For best transfer results, use the highest electric field strength (E) possible within the heat dissipation capabilities of the system. For most proteins, the most rapid transfer occurs under conditions where the applied voltage (V) is maximized and the distance between the electrodes is minimized. Though rapid blotting experiments may seem to be the most convenient, a number of factors must be considered when choosing the appropriate power conditions for a given electrophoretic transfer.
Two basic equations are important in electrophoresis. The first is Ohm's law, which relates the applied voltage (V) with the current (I) and resistance (R) of the system:
V = I x R
The applied voltage and current are determined by the user and the power supply settings; the resistance is inherent in the system.
The second equation, the power equation, describes the power (P) used by a system, which is proportional to the voltage (V), current (I), and resistance (R) of the system:
P= I x V = I2 x R = V2/R
Understanding the relationships among power, voltage, current, resistance, and heat is central to understanding the factors that influence the efficiency and efficacy of transfer.
The power that is dissipated is also equivalent to the amount of heat, known as Joule heating, generated by the system. According to the power equation, the amount of Joule heating that occurs depends on the conductivity of the transfer buffer used, the magnitude of the applied field, and the total resistance within the transfer system. During an electrophoretic transfer, the transfer buffer warms as a result of Joule heating, and consequently, resistance drops. Such heating and changes in resistance may lead to inconsistent field strength and transfer, may cause the transfer buffer to lose its buffering capacity, or may cause the gel to melt and stick to the membrane. Under normal running conditions, the transfer buffer absorbs most of the heat that is generated; during extended transfer periods or high-power conditions, active buffer cooling is required to prevent uncontrolled temperature increases.
The following variables also change the resistance of the transfer system and, therefore, also affect transfer efficiency and current and voltage readings:
In theory, increasing the power input and duration of an electrophoretic transfer results in the transfer of more protein out of a gel. In practice, however, test runs should be used to evaluate transfer efficiency at various field strengths (by modulating both power input and, if applicable, interelectrode distance) and transfer times for each set of proteins of interest. The optimum transfer conditions depend on a number of factors, including the size, charge, and electrophoretic mobility of the protein, the type of transfer buffer used, and the type of transfer system being used.
As their name suggests, high-intensity field transfers use high-strength electrical fields that are generated by increased voltage and closer positioning of electrodes. High-intensity transfers often produce satisfactory transfer of proteins in less time than standard transfers; however, in some cases the high field strength causes small proteins to be transferred through the membrane (protein blow-through). In addition, high molecular weight proteins and other proteins that are difficult to transfer may not have sufficient time to be transferred completely. Since more heat is generated in high-intensity field transfers than in standard field transfers, a cooling device may be needed.
Standard field transfers require less power input and more time to complete, so they are generally run overnight. Standard transfers often produce more complete, quantitative transfer of proteins across a broad molecular weight range; the slower transfer conditions allow large proteins sufficient time to move through the gel matrix while the lower intensity allows smaller proteins to remain attached to the membrane after transfer.
Tank transfer systems offer the capacity for both high-intensity and standard-field transfers. Increased buffering capacity and additional cooling mechanisms enable longer transfer times than are feasible with semi-dry transfers. Some tank transfer systems offer flexible electrode positions that, when combined with variable voltages, provide a choice of high-intensity, rapid transfer or longer, more quantitative transfer over a broad range of molecular weights. Semi-dry transfers, on the other hand, are necessarily rapid and of high intensity. In a semi-dry transfer system, the distance between electrodes is determined only by the thickness of the gel-membrane sandwich, and buffering and cooling capacity is limited to the buffer in the filter paper. As a result, the field strength is maximized in semi-dry systems, and the limited buffering and cooling capacity restricts the transfer time. Though power conditions may be varied with the power supply, semi-dry transfers often operate best within a narrow range of settings.
If the voltage is held constant throughout a transfer, the current in most transfer systems increases as the resistance drops due to heating; the exception is most semi-dry systems, where current actually drops as a result of buffer depletion. Therefore, the overall power increases during transfer, and more heating occurs. Despite the increased risk of heating, a constant voltage ensures that field strength remains constant, providing the most efficient transfer possible for tank blotting methods. Use of the cooling elements available with the various tank blotting systems should prevent problems with heating.
If the current is held constant during a run, a decrease in resistance results in a decrease in voltage and power over time. Though heating is minimized, proteins are transferred more slowly due to decreased field strength.
If the power is held constant during a transfer, changes in resistance result in increases in current, but to a lesser degree than when voltage is held constant. Constant power is an alternative to constant current for regulating heat production during transfer.
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