The Electric Field
The transfer of proteins from gel to membrane is accomplished via an electric field. This field is dependent on Ohm’s law, which relates voltage (V), current (I), and resistance (R) as V = I × 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 between power, voltage, current, resistance, and heat is critical to achieving a great Western blot transfer
The goal is to produce a high electric field measured in volts/cm of membrane without producing excessive amounts of heat. Heat generation during electrotransfer is undesirable as it will cause elevated background on your blot, this is especially challenging for fluorescent Westerns. At Advansta we use the Genie® Electrophoretic Transfer system from iDEA Scientific for wet transfers. We love this system because it uses plate electrodes to create a higher strength electric field and greater current density than conventional wire-electrode systems. Using a constant low voltage and high current produces a constant field strength that yields high transfer efficiency.
The Transfer Buffer
The electric field is conducted via a transfer buffer. The buffer’s job is to maintain a stable pH, drive the elution of proteins from the gel and adhere them to the membrane. Typical buffer systems include Tris/Glycine or CAPS with the addition of alcohol and/or detergent depending on the properties of the target protein. When it comes to transfer efficiency, choosing the right transfer buffer is just as important as selecting the transfer apparatus.
Advansta offers a novel transfer buffer, FLASHBlot Transfer Buffer, which enhances the transfer of proteins from electrophoretic gels to membranes for immunoblotting. Compared to commonly used transfer buffers, FLASHBlot Transfer Buffer provides improved transfer efficiency of all proteins, including those of high molecular weight. FLASHBlot Transfer Buffer also allows for increased protein retention on membranes resulting in more sensitive detection of low-abundance proteins. Transfers with the FLASHBlot buffer can be performed with all traditional wet transfer apparatuses. The decreased transfer times plus increased transfer efficiency and protein retention allow for sensitive detection of post-translationally modified proteins, which can often be difficult to detect after traditional transfer protocols.
FlashBlot Transfer Buffer