Abstract
A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 401-408 |
| Number of pages | 8 |
| Journal | Analytical Biochemistry |
| Volume | 135 |
| Issue number | 2 |
| DOIs | |
| State | Published - Dec 1983 |
Bibliographical note
Funding Information:We thank Mr. Robert Petersen for providing us with micrococcal nuclease-treated lysates of rabbit reticulocytes and discussions throughout this project. We express our appreciation to Dr. Fred Forro for providing us with many items of equipment used in these experiments. This work was supported by a grant from the National Science Foundation to PBH.
