By John M. Walker
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Additional info for New Nucleic Acid Techniques (Methods in Molecular Biology Vol 4)
D. (1975) Quantitative and “%I in polyacrylamide gels by fluorography. 5-341. film detection of 3H Eur. 1. Biochem. 56, Chapter5 In Vitro Translation and Analysis of Early Events in Protein Synthesis Initiation in Nonreticulocyte MammalianCells Jeffrey IM Pollard and Michael J. Clemens 1. Introduction The most commonly used systems to translate mRNAs in vitro are the rabbit reticulocyte lysate and the wheat germ extract (2,2 andsee Vol. 2 in this series). Both these systems have a number of advantages, including the ease and cheapness of preparation, the relative absence of RNAse, the high level of reinitiation of ribosomes onto mRNA, the high fidelity of translation, and themaintenance of activity uponlong-term storage.
8. 9. 10. 11. 12. 13. 14. 15. Slater Parnes, J. , and Seidman, J. G. (1981) Mouse p2 microglobulin cDNA clones: A screening procedure for cDNA clones corresponding to rare mRNAs. Proc. Natl. Acad. Sci. USA 78,2253-2257. Fischer, S. G. (1983) Peptide mapping in gels. M&h. Enzymol. 100,424430. , and Tucker, G. A. (1985) The appearance of polygalacturonase mRNA in tomatoes: One of a series of changes in gene expression during development and ripening. Planta 163,263-271. Jackson, R. C. and Blobel, G.
Notes 1. 2. 3. 4. It is essential that cells are in the exponential phase of growth when they are harvested because the rate of protein synthesis rapidly drops either as the cells enter stationary phase at high density or as the result of nutrient deprivation. The gel-filtered lysate is sometimes used in order to have more defined components and higher specific activities of radioactive amino acids. The procedure results, however, in a 2-3-fold dilution and a substantial loss of activity. Endogenous energy sources are also removed by this procedure and may need to be compensated for.
New Nucleic Acid Techniques (Methods in Molecular Biology Vol 4) by John M. Walker