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During electrophoresis, negatively charged proteins migrate from the gel towards the anode, contacting and binding to the membrane One gel is transferred per module, and two modules fit in one tank. Transfer speed ranges from 1 h to overnight, usually at a constant voltage to maintain field strength and maximize efficiency of transfer.
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Learn methods on how to efficiently transfer proteins from a gel to a membrane for maximum western blot sensitivity. Make sure the cassette clamps are removed before you set up your transfers This article reviews and compares transfer methods, addresses the properties of membranes and why to choose one over another, and provides recipes for the various transfer buffers used in western blot transfer.
The polyacrylamide gel and transfer membrane are placed in direct contact before being sandwiched between a negative electrode (cathode) and a positive electrode (anode)
The cathode is placed behind the gel, and the anode is placed behind the membrane. Wet transfer is less prone to failure due to drying of the membrane, and is especially recommended for large proteins For both kinds of transfer, the membrane is placed next to the gel. The membrane should be closest to the anode (+) as the protein with negative charge would move towards the anode
Connect the transfer tank to the power supply and transfer according to the recommendations of the manufacturer. Transfer of proteins from gel to membrane The gel is placed in contact with the membrane and the proteins then migrate toward the positively charged anode (+) in an electric field The electric field is fundamental for successful protein transfer.
Place the cassette in the transfer tank with the correct electrode orientation (cathode and anode), ensuring complete immersion in the transfer buffer
This section provides an overview of the transfer conditions required for performing electrophoretic protein transfer It also provides some troubleshooting tips for electrophoretic transfer and outlines a general workflow for the protein transfer from gels to membranes. In this video, you’ll learn how to perform a western transfer using the mini blot module in a mini gel tank
