Elution of polypeptides from the gel

Proteins may be eluted from gels by diffusion, solvent convection or electro-elution. 

Diffusion is simple but rather slow and resolution may be poor compared to the other methods. The gel is placed between two sheets of nitrocellulose membranes, which are in turn inserted between two filter papers covered with foam pads and rigid screens (Bowen et al., 1980 Aubertin et al., 1983 Table 16.9). High-molecular weight polypeptides are transferred less efficiently in the presence of SDS. Nevertheless, in the presence of SDS more faithful replicas 

Diffusion-driven transfer in the absence ofSDS (Bowen et al., 1980) 

Immerse the gel after electrophoresis in 10 mM Tris-HCl buffer, pH 7.0, containing 2 mM EDTA and 50 mM NaCI (transfer buffer) for 3 h. 

Place the gel between 2 sheets of welted nitrocellulose membrane (pore size 0.45 /im or, preferably, 0.2 m), avoiding any air bubbles. Cover each membrane with filter paper, foam pad and rigid screen, all previously wetted and attach with rubber bands. 

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