Estimating the amounts of ADP-ribosylatable active elongation factor-2 in mammalian cell-free extracts

The content of the elongation factor (EF-2) can be measured by diphtheria toxin-dependent ADP-ribosylation in cell-free extracts of samples prepared from small amounts of tissues and cells containing less than 100 micrograms of total protein. A 20 min in vitro assay, in which a radioactive ADP-ribosyl residue is transferred specifically and 1:1 stoichiometrically to EF-2, is sufficient to estimate the total amounts of ADP-ribosylatable active EF-2. The method is very useful for monitoring changing levels of EF-2 during various pathological and biological processes, including cell cycle, ageing, cancer and other diseases.