Multiple gels may be electrotransferred in the standard field option, which is performed either at constant current (0.1 to 1 A) or voltage (5 to 30 V) from as little as 1 hour to overnight. The supported gel sandwich is placed vertically in a tank between stainless steel/platinum wire electrodes and the tank is filled with transfer buffer. The gel is then placed in the “transfer sandwich” (filter paper-gel-membrane-filter paper), cushioned by pads and pressed together by a support grid. When performing a wet transfer, the gel is first equilibrated in transfer buffer. The efficiency of protein transfer can be affected by the chemistry, thickness of the gel, the molecular weight of the proteins being transferred, the type of membrane and transfer buffers used, and the transfer method. In addition to the challenges of immunodetection in the protein blotting workflow, the transfer of proteins from a gel matrix to a membrane is a potential hurdle. An appropriate method is then used to detect the localized probe to document the location and relative abundance of the target protein. The transferred protein is then probed sequentially with antibodies and detection probe (e.g., enzyme, fluorophore, isotope).
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