Measuring the Affinity of the Bacterial Rho Helicase for RNA Using a Double-Membrane Slot-Blot Filtration Assay.

Quantitating the binding of oligomeric proteins to RNA is not always straightforward with standard biochemical approaches such as the Electrophoretic Mobility Shift Assay (EMSA). Here, we detail how we use a simple, alternative approach based on sample filtering through stacked nitrocellulose and positively charged nylon membranes to measure the affinity of the hexameric transcription termination factor Rho for RNA. The method is quick and easy to implement and should be useful to study the interaction of other RNA-binding proteins using RNA substrates bearing a 32P or fluorescent label.