The technique involves sandwiching a single gel between two membranes and clamping it between glass plates to facilitate diffusion. The diffusion method is not widely utilized as it provides qualitative rather than quantitative transfer, but is especially useful in producing up to 12 blots per SDS-PAGE gel for screening with multiple antibodies. PVDF is also physically stronger than nitrocellulose, so may be preferable for stripping and reprobing. PVDF offers a higher binding capacity than nitrocellulose, but likewise has higher background binding. A number of membranes have been developed in the past, but only nitrocellulose and polyvinylidene fluoride (PVDF) remain popular. Effective protein transfer is also heavily reliant on the gel acrylamide percentage, the molecular weight of electrophoresed proteins, and the blotting membrane used. Electroelution (wet, semi-dry, and dry) and diffusion transfer techniques all have their merits and applications, but it is important to note that method alone will not guarantee a successful blot. There are many safer methods for transferring proteins to a membrane. The protein transfer stage should carry equal risks to PAGE, although many labs adopt the method depicted in Figure 1 of the aforementioned article, which introduces further possibility of electric shock. The buffer should be covered at all times when attached to the power supply to prevent contact with the electrified liquid. Some tanks allow the unit to be plugged into the power pack without covering the buffer. Polyacrylamide gel electrophoresis (PAGE) is only unsafe if the equipment is not used correctly. To limit risk they should be handled with care, in a fume hood with the use of personal protective equipment. Protein extraction often uses β-mercaptoethanol and sodium dodecyl sulfate (SDS), both of which are corrosive, acutely toxic and the former mutagenic. Figure 7 from ‘Western Blot: Technique, Theory, and Trouble Shooting’.Īlthough Western blotting involves passing an electric current through a liquid environment, it is not a dangerous technique if performed properly however, it is easy to overlook the risks of each stage.
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