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. 1985 Mar 1;226(2):361–368. doi: 10.1042/bj2260361

Analytical errors in measuring radioactivity in cell proteins and their effect on estimates of protein turnover in L cells.

J A Silverman, J Mehta, S Brocher, J S Amenta
PMCID: PMC1144720  PMID: 3994661

Abstract

Previous studies from this laboratory on protein turnover in 3H-labelled L-cell cultures have shown recovery of total 3H at the end of a 3-day experiment to be always significantly in excess of the 3H recovered at the beginning of the experiment. In this study we have critically reviewed a number of possible sources for this error in measuring radioactivity in cell proteins. 3H-labelled proteins, when dissolved in 0.3 M-NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, approx. 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. To aggravate this latter loss further, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Airhart J., Arnold J. A., Bulman C. A., Low R. B. Protein synthesis in pulmonary alveolar macrophages. Source of amino acids for leucyl-tRNA. Biochim Biophys Acta. 1981 Mar 26;653(1):108–117. doi: 10.1016/0005-2787(81)90109-x. [DOI] [PubMed] [Google Scholar]
  2. Amenta J. S., Sargus M. J. Mechanisms of protein degradation in growing and non-growing L-cell cultures. Biochem J. 1979 Sep 15;182(3):847–859. doi: 10.1042/bj1820847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boer G. J. A simplified microassay of DNA and RNA using ethidium bromide. Anal Biochem. 1975 May 12;65(1-2):225–231. doi: 10.1016/0003-2697(75)90507-2. [DOI] [PubMed] [Google Scholar]
  4. Brown W. O. Liquid-scintillation counting of C-labelled animal tissues at high efficiency. Biochem J. 1961 Mar;78(3):571–578. doi: 10.1042/bj0780571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DULBECCO R., VOGT M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 1954 Feb;99(2):167–182. doi: 10.1084/jem.99.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. HERBERG R. J. Phosphorescence in liquid scintillation counting of proteins. Science. 1958 Jul 25;128(3317):199–200. doi: 10.1126/science.128.3317.199. [DOI] [PubMed] [Google Scholar]
  7. Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
  8. Hildebran J. N., Airhart J., Stirewalt W. S., Low R. B. Prolyl-tRNA-based rates of protein and collagen synthesis in human lung fibroblasts. Biochem J. 1981 Aug 15;198(2):249–258. doi: 10.1042/bj1980249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Schneible P. A., Airhart J., Low R. B. Differential compartmentation of leucine for oxidation and for protein synthesis in cultured skeletal muscle. J Biol Chem. 1981 May 25;256(10):4888–4894. [PubMed] [Google Scholar]
  10. Silverman J. A., Amenta J. S. Sources of error in estimating radioactivity in protein from cell cultures by liquid scintillation counting. Anal Biochem. 1984 Sep;141(2):538–544. doi: 10.1016/0003-2697(84)90082-4. [DOI] [PubMed] [Google Scholar]
  11. VAUGHAN M., STEINBERG D., LOGAN J. Liquid scintillation counting of C14- and H3-labeled amino acids and proteins. Science. 1957 Sep 6;126(3271):446–447. doi: 10.1126/science.126.3271.446-a. [DOI] [PubMed] [Google Scholar]
  12. Wallyn C. S., Vidrich A., Airhart J., Khairallah E. A. Analysis of the specific radioactivity of valine isolated from aminoacyl-transfer ribonucleic acid of rat liver. Biochem J. 1974 Jun;140(3):545–548. doi: 10.1042/bj1400545. [DOI] [PMC free article] [PubMed] [Google Scholar]

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