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from Promega. Labeled riboprobes are transcribed by inclusion of [a- P]UTP (800 Ci/mmol; NEN Life Science Products, Boston, MA) in the transcription reaction. Reaction mixtures are treated with 1 U of DNase I and riboprobes are purified with the Promega Wizard PCR Prep DNA cleanup system. Bound transcripts are eluted with RNase-free water and stored at —20° in the presence of RNasin (1 U//il; Promega).

Radiolabeled riboprobe (1 x 105 cpm) is incubated with 15-20 of protein extract in buffer containing 12 mM HEPES (pH 7.9), 15 mM KC1, 5 mM MgCl2, 2 mMdithiotreitol (DTT), 1 fig of tRNA, heparin (1 fig/fil), RNasin (1 U/^l), and

17 J. Falck, P. Dagger, B. Jensen, and M. Sehested, J. Biol. Chem. 274, 18753 (1999).

10% (v/v) glycerol in a total volume of 20 fi\ for 20 min at 25°. Total cellular protein extracts are prepared by repeated freeze-thawing in buffer containing 10 mM HEPES, 1.5 mM MgCl2, 1 mM EDTA, 0.2 mM EGTA, 10 mM KC1, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10% (v/v) glycerol. For competition experiments, the protein extract is first incubated for 10 min at 25° with unlabeled competitor RNA (specific or nonspecific) before the addition of the radiolabeled transcript. Control reactions without competitors are sham treated under identical conditions. RNA-protein binding reactions are treated with 5 U of RNase T1 for 15 min at 25° and separated by electrophoresis on a 4.5% (w/v) native polyacrylamide gel in 45 mM Tris, 45 mM boric acid, and 1.2 mM EDTA buffer, pH 7.4. Radioactive bands are visualized by exposing the dried gels to a Phosphorlmager screen.

Results presented in Fig. 3 show that the mobility of the 177-nt Topo II3' UTR transcript is retarded in the presence of protein extract compared with the control reaction without protein extract (compare lanes 1 and 2 in Fig. 3). The retardation

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