Selected article for: "bicistronic reporter and FLuc RLuc ratio"
Author: Wang, Qing S.; Jan, Eric
Title: Switch from Cap- to Factorless IRES-Dependent 0 and +1 Frame Translation during Cellular Stress and Dicistrovirus Infection
  • Document date: 2014_8_4
    • ID: 0if5z3xp_13
    Snippet: In a 10 ml reaction, either 1 mg linearized bicistronic reporter constructs or 500 ng of capped reporter RNAs were incubated with 6.7 ml of Sf21 cell extract (Promega), 0.3 ml L-[ 35 S]methionine (PerkinElmer, .1000 Ci/mmol), 40 mM KOAc and 0.5 mM MgCl 2 at 30uC for 1.5-2 hours as indicated [31] . Capped RNAs were preheated at 65uC for 3 minutes and incubated in buffer E (20 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgOAc, 0.25 mM Spermidine, and 2 mM D.....
    Document: In a 10 ml reaction, either 1 mg linearized bicistronic reporter constructs or 500 ng of capped reporter RNAs were incubated with 6.7 ml of Sf21 cell extract (Promega), 0.3 ml L-[ 35 S]methionine (PerkinElmer, .1000 Ci/mmol), 40 mM KOAc and 0.5 mM MgCl 2 at 30uC for 1.5-2 hours as indicated [31] . Capped RNAs were preheated at 65uC for 3 minutes and incubated in buffer E (20 mM Tris pH 7.5, 100 mM KCl, 2.5 mM MgOAc, 0.25 mM Spermidine, and 2 mM DTT) at room temperature for 10 min before adding to the reaction. Proteins were separated by a 16% SDS polyacrylamide gel, exposed to a phosphor storage screen (Molecular Dynamic) and quantified using a Typhoon imager 8600 (Amersham). When calculating the ratio of FLuc/ RLuc by [ 35 S]-methionine labeling, the number of methionines were taken into account of each reporter protein.

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