Often, the number of ribosomes which undergo À1 frameshift has been determined from constructs such as our 'Upstream stop' constructs, by separating radioactively labelled proteins by SDS-PAGE and quantifying the relative amount of protein in each of the two polypeptides (28 versus 148 kDa). Given the limited resolution of SDS-PAGE, it is, however, impossible to clearly differentiate between polypeptides produced by ribosomes that terminate at the in-frame UAA stop codon and ribosomes that undergo À1 frameshift but stall within the pseudoknot. In order to overcome this problem, we invoked 2D SDS-PAGE (34) whereby polypeptides were separated not only by molecular weight but also by their isoelectric point (pI).