Sources of error in estimating radioactivity in protein from cell cultures by liquid scintillation counting

Abstract

In this study, several common sample preparation techniques for liquid scintillation counting were critically reviewed. It has been shown that techniques such as NaOH or formic acid solubilization of trichloroacetic acid (TCA)-precipitated proteins led to underestimation of the radioactivity by 20–40%; this loss was not corrected by either internal or external standardization. Hydrolysis of the proteins with 6 n HCl or Pronase significantly increased the recovery of the labeled proteins. Also, 10% of the labeled cell proteins remained on the dish when cells were scraped into buffer; these labelled proteins could be recovered either by in situ hydrolysis with Pronase or by solubilization and scraping in 0.3 n NaOH. These techniques increased the recovered radioactivity by 50–60%, allowing quantitative measurements to be made over a 3-day chase period. A possible mechanism and the implications of this observation were discussed.