Comparison of transfer methods in Western blottingAfter gel electrophoresis, Western blotting starts with the transfer of proteins from the gel to membrane. Electrotransfer is a common technique for step and takes advantage of the same electromobility principles as gel electrophoresis.Wet and semidry transfer are the two most common electrotransfer methods and provide greater speed and efficiency than alternatives based on diffusion, capillary action, and vacuum. What is wet transfer?Wet transfer is generally suitable for all proteins and uses a cassette that sandwiches foam pads, filter paper, gel, and a membrane together.
The cassette is submerged in transfer buffer and orientated with the membrane on the anode side (Fig 1), to ensure proteins move in the correct direction.During electrophoresis, negatively charged proteins migrate from the gel towards the anode, contacting and binding to the membrane. Transfer speed ranges from 1 h to overnight, usually at a constant voltage to maintain field strength and maximize efficiency of transfer.Long transfer times at a constant voltage lead to unwanted heat, decreasing resistance, and increasing current. Adequate cooling measures to prevent buffer breakdown are often required, including chilled buffers, ice packs, and performing transfers in a cold room at 4°C.
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Some wet transfer systems use integrated cooling.Fig 1. The wet transfer sandwich is assembled (top), placed in a cassette, and submerged in a transfer tank filled with buffer with membrane closest to the anode (bottom). What is semidry transfer?Semidry transfer follows the same principles as wet transfer but is faster and uses smaller volumes of buffer.The gel and membrane are sandwiched between sheets of filter paper soaked in transfer buffer, with electrode plates on either side (Fig 2). As in wet transfer, proteins move out of the gel toward the anode when an electric field is applied, binding to the membrane.Unlike wet transfer, semidry transfer uses minimal buffer and requires minutes, compared to hours. The electrode plates absorb heat during the short run time, necessitating fewer cooling measures. However, semidry transfer is less efficient overall, noticeably affecting the transfer of high molecular weight proteins.Fig 2. Construction of a gel-membrane sandwich in a semidry transfer setup is similar to wet transfer.
Western blotting, the transfer of proteins to a solid-phase membrane support followed by immunodetection, is a powerful and popular technique for the visualization and identification of proteins. Western blotting combines the resolution of gel electrophoresis with the specificity of immunoassays, allowing the identification and analysis of. Protein transfer is a vital step in Western blotting involving the transfer of proteins separated in a gel by electrophoresis to a solid support matrix so that specific proteins can be detected using immunodetection techniques. We offer a complete array of products to support rapid and efficient protein transfer for Western blotting.
All components are soaked in transfer buffer and placed in direct contact with the electrodes, compressing the sandwich.Following either wet or semidry transfer, the membrane can be stained or immunoblotted, as needed. What are the advantages/disadvantages of these methods?Table 1 summarizes the main differences between wet and semidry transfer.Table 1. Comparison of wet and semidry transfer of proteins. AdvantagesDisadvantagesWet transferEfficient transfer for small and large proteinsRequires adequate cooling measuresClear, sharp bands on membraneLarge volumes of bufferParticularly suited for transfer of large proteinsRunning times from 1 h to overnightSemidry transferShort run times (.