Binding and Washing Conditions

The conditions used for binding displayed protein to RNA depend on the protein studied. In the case of U1A, salt and buffer conditions were based on published work (Hall and Stump, 1992; Scherly et ai, 1990). Magnesium was omitted from the binding buffer to minimize possible RNase activity, and a litde detergent (0.5% Triton X-100) was added to reduce background binding. Gel shift analyses showed that these modifications did not affect the ability of U1A to bind tighdy to the UlhpII target RNA. Ideally, the capacity of a protein to bind to its RNA target in the display binding buffer should always be tested beforehand.

Once RNA-bound phage is trapped on the beads, the free phage must be washed away. We use an excess of binding buffer for this purpose. Since the bound phage can be released during washing, it is important to wash as quickly as possible. This is facilitated by the paramagnetic beads, which can be pulled to the side of a microfuge tube within 30—60 sec. Extended washing steps can be used to select for protein variants that release their RNA target very slowly.

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