A rapid and accurate procedure for assaying DNA polymerase, RNA polymerase, or ribosome dependent protein synthesis
✍ Scribed by Bernard I. Weinstein; Nina Bhardwaj; Heng-Chun Li
- Publisher
- Elsevier Science
- Year
- 1975
- Tongue
- English
- Weight
- 384 KB
- Volume
- 68
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A descending paper chromatographic procedure for separating trichloracetic acid soluble from precipitable material is described. Its use for assaying DNA, RNA, or protein synthesis from a variety of sources with various states of purity attests to its general applicability to reactions of interest to molecular biologists. Large numbers of analyses can be carried out in a short period of time without compromising accuracy or reliability. The use of this procedure for still more enzymes, those involved in modifying DNA, RNA, or proteins, is discussed.
In the course of our experiments on RNA synthesis utilizing chromatin as a template it became obvious that a better method of separating the radioactively labeled RNA product from the precursor nucleotides would be quite valuable. The standard trichloracetic acid (TCA) precipitation followed by either centrifugation or filtration is very laborious and time consuming when large numbers of samples are to be analyzed. Several methods have been described in which the reaction mixture is absorbed onto paper disks, then the product is precipitated and washed free of precursor in a large batch. In this manner assays have been described for DNA polymerase (i), protein kinase (2), and UDP glucose-Glycogen Glucosyltransferase (3). This batch washing procedure was later generalized for RNA polymerase and protein synthesis (4). More recently a descending paper chromatography method has been described which requires fewer manipulations and more efficiently separates unincorporated precursor from the reaction product for protein kinase (5), polyphosphate kinase (6), and for phosphoprotein phosphatase (7). We now report on the adaptation of the descending paper chromatographic procedure so that it can be conveniently and reliably used to measure incorporation of radioactive precursors into DNA, RNA, or protein.
METHODS
Trichloroacetic acid precipitation and chromatographic development.
Whatman 31 ET chromatography paper was cut into sheets of approxi-62