Translation Initiation Extract Systems and Molecular by JOHNN.ABELSON AND MELVINI.SIMON

By JOHNN.ABELSON AND MELVINI.SIMON

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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.

1A, the exact amount will vary depending on the mRNA [in part as the 50 and 30 untranslated regions (UTRs) and in part the coding region]. We have no theoretical explanation for the differences, but the differences are quite real and very reproducible. 0 mg per reaction. 1A and B shows the best, worst, and average mRNAs from a much larger number of mRNAs that we have studied. The second variable to optimize is time. Depending on the mRNA in use, we have found that the optimal time varies from 20 to 60 min.

8 William C. Merrick and Diane Barth-Baus Running buffer: 1. Tris base 2. Glycine 3. SDS 4. 9 liters 10 liters The advantage of using gel electrophoresis is that the protein band in question is readily seen and possible evaluation of ‘‘side products’’ is easily achieved (something not observable with hot TCA-precipitable radioactivity). The disadvantage is the lack of absolute, quantitative control. This disadvantage can be corrected for by the use of a reliable mRNA included in the series as an internal control for the gels to be run.

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