![]() ![]() Transfer efficiency increases with increasing transfer time. Transfer efficiencies of 80-100% can be achieved for proteins between 14-116 kDa. Scheme showing the assembly of a typical western blot apparatus with the position of the gel, the transfer membrane, and the protein orientation relative to the electrode position. Tank transfer apparatus for western blotting. A high field option is available that can reduce the transfer time to 30 minutes for a single gel, but requires the use of high voltage (up to 200 V) or high current and a cooling system to dissipate the enormous heat generated. Transfers are typically performed with an ice pack and at 4☌ to reduce the heat generated. Multiple gels can be electrotransferred in the standard field option, which is performed at constant current (0.1 to 1 A) or voltage (5 to 30 V) for as little as 1 hour to overnight. The supported gel sandwich is placed vertically in a tank between the stainless steel/platinum wire electrodes and filled with transfer buffer. The gel is then placed in a "transfer sandwich" (filter paper-gel-membrane-filter paper), padded with pads and pressed together by a support grid. When wet transfer is performed, the gel is first equilibrated in the transfer buffer. Wet Tank Electrotransfer (Wet Tank Transfer) Three main ways to manufacture proteins from SDS-PAGE or natural gels to nitrocellulose, PVDF or nylon membranes: Dry electroblotting offers high quality transfer with both speed and convenience as no additional buffers are required for dry electroblotting. However, semi-dry transfer may have a lower efficiency in transferring large molecular weight proteins (>300 kDa). Semi-dry drying provides convenience and time savings with the flexibility to use multiple buffer systems or preassembled batches without buffers. Traditional wet transfer offers high efficiency, but at the expense of time and effort. The accuracy of the results depends on the transfer efficiency of the western blot method. Western Blot Wet, Semi-Dry and Dry Transfer Methods (Wet Transfer, Semi-Dry Transfer, Dry Transfer)Įfficient and reliable protein transfer from the gel to the blotting membrane is the cornerstone of a successful western detection experiment. ![]()
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