Simultaneous preparation of nuclear DNA, RNA, and protein from carcinogen treated-hamster embryo fibroblasts
✍ Scribed by Michael C. MacLeod; Betty K. Mansfield; Anne Huff; James K. Selkirk
- Publisher
- Elsevier Science
- Year
- 1979
- Tongue
- English
- Weight
- 888 KB
- Volume
- 97
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
Nuclei from hamster embryo fibroblasts treated with radioactive benzo(a)pyrene were lysed in 6 M guanidine, and nuclear macromolecules were separated by isopycnic centrifugation in CslSOI. Control experiments showed that cross-contamination of the RNA, DNA, and protein fractions was less than 2% of the total recovery of each macromolecular class. When compared to previous techniques utilizing phenol extraction, similar specific activities of bound hydrocarbon (pmol benzo[a]pyrene/mg protein or nucleic acid) were obtained. However, overall recoveries of macromolecular components were higher with the present method. In addition, recovery of undegraded histones in the density gradient preparation of nuclear protein was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and recovery of native DNA was demonstrated by thermal denaturation studies. Although developed specifically for work with carcinogenic hydrocarbons, the Cs$O, technique should be generally useful in cases where it is necessary to prepare all three classes of macromolecules from one batch of nuclei Carcinogenic, polycyclic aromatic hydrocarbons such as benzo( a)pyrene [B( a)P14 are metabolized by mammalian cells (1,2) to activated intermediates which react with all major classes of cellular macromolecules (3-5), in addition to a number of small I The U. S. Government's right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged.