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Protein transfer in tobacco smoke has been studied using the protease, Savinase™, as a model protein. Mainstream and sidestream smoke were collected from cigarettes to which Savinase had been added at various concentrations. Savinase was extracted from the smoke condensate with an organic solvent system before being precipitated and further identified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. The detection limit of the method, based on addition of Savinase to the smoke condensate, was 25 µg in mainstream and 100 µg in sidestream smoke. At a Savinase concentration of 6000 µg per gram of tobacco, the methodology allows the detection of protein transfer as low as 0.009% and 0.054% in mainstream and sidestream smoke, respectively. Using this approach, it was shown that there is no detectable Savinase in the mainstream and sidestream smoke of filtered and unfiltered cigarettes containing up to 6000 µg of Savinase per gram of tobacco. These facts strongly suggest that there is no significant transfer of protein from tobacco into cigarette smoke.