Quantitative Film Detection of 3H and 14C in Polyacrylamide Gels by Fluorography

TLDR

Fluorography using PPO scintillator has been previously described, and recent revisions extend the method to agarose gels and include a rapid PPO recovery procedure. The study shows that briefly pre‑exposing film to light markedly boosts fluorography sensitivity. Pre‑exposure linearizes the film response, enabling quantitative micro‑densitometry of radioactivity. Micro‑densitometry of pre‑exposed film accurately reflects radioactivity distribution, detecting as low as 300 dis.

Abstract

Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by micro-densitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.