By JOHNN.ABELSON AND MELVINI.SIMON
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Extra info for Translation Initiation Extract Systems and Molecular Genetics
Translation Initiation in Reticulocyte Lysates 3 This chapter will cover the general conditions for the use of nuclease-treated rabbit reticulocyte lysates, optimization, types of reporters, basic experiments and then more refined experiments that vary the standard conditions for mRNA utilization, and the influence of changing the effective activity for various translation initiation factors (either by the addition of exogenous initiation factors or by the direct inhibition of endogenous initiation factors).
1981; Translation Initiation in Reticulocyte Lysates 13 Godefroy and Thach, 1981). Starting with a mixture that is roughly one to one on a molar basis for each mRNA, the mixture is titrated into the lysate until well past saturation (Fig. 2A). What is observed in this process is the following. At limiting mRNA concentrations, the proteins expressed are roughly in proportion to the amount of mRNA present (as noted above, in the bicistronic mRNA, IRES-mediated translation is artificially suppressed).
After the sample has been applied, it is washed twice with 2 ml of cold 10% TCA and then finally with 2 ml of cold 95% ethanol. The ethanol wash removes the last traces of TCA; failure to do so may result in some quenching when the samples are subjected to liquid scintillation spectrometry. The filters are then dried for 10 min under a heat lamp, placed in scintillation vials, and scintillation cocktail is added. Radioactivity is then determined using scintillation spectroscopy. The advantage of this procedure is that it is relatively rapid and very quantitative.