![]() Soak filter paper in semi-dry or tank-blotting transfer buffer.Incubate membrane for 10 min in semi-dry or tank-blotting transfer buffer.Tip: To avoid contamination, always handle the filter paper, membrane, and gel with gloves. Cut 8 pieces of filter paper and a piece of membrane to the same size as the gel.Once transferred to the membrane, the proteins can be probed with epitope-specific antibodies or conjugates. Transfer efficiency can be checked by staining proteins on the membrane using Ponceau S (see Ponceau S staining). Results obtained with the tank-blotting method are typically better, with more efficient transfer, particularly of large proteins. With the tank-blotting method, a blotting cassette is submerged in a tank for blotting (see figure Tank- and semi-dry blotting methods).Tank blotting can be performed over extended periods since the buffer capacity is far greater than that with semi-dry transfer systems. SDS-coated, negatively charged proteins are transferred to the membrane when an electric current is applied. The membrane is placed near the anode (positively charged), and the gel is placed near the cathode (negatively charged). With the semi-dry electroblotting method, the gel and membrane are sandwiched between two stacks of filter paper that have been pre-wet with transfer buffer. Following electrophoresis, proteins in a polyacrylamide gel can be transferred to a positively charged membrane (e.g., Schleicher and Schuell BA85) in a buffer-tank–blotting apparatus or by semi-dry electroblotting as described below. ![]()
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