The protein synthesis inhibition activity of the recombinant OsRIP1 and controls (DPBS buffer and BSA) was determined using an in vitro transcription/translation system [49 (link)]. To test the effect of OsRIP1 on animal ribosomes the TnT® T7 Quick Coupled Transcription/Translation System Kit (Promega, Mannheim, Germany) based
On a cell-free system derived from rabbit reticulocytes. The effect on plant ribosomes was analyzed using the TnT® T7 Coupled Wheat Germ Extract kit (Promega) was used. According to the manufacturer’s instructions, the prepared mixture was incubated at 30°C for 10 min and chilled on ice. Afterwards, 2 µL DPBS buffer or buffer containing different concentrations of OsRIP1 was added to the reaction mixture and incubated for 30 min at 30 °C. After addition of 35 µL nuclease-free water at room temperature, the reaction samples were transferred to a luminometer plate (Greiner Labortechnik, Frickenhausen, Germany) containing 5 µL luciferase assay reagent at 25 °C. The relative luciferase activities of the samples were determined using a microtiter top plate reader (Infinite F200 Pro, Tecan, Mannedorf, Switzerland).
On a cell-free system derived from rabbit reticulocytes. The effect on plant ribosomes was analyzed using the TnT® T7 Coupled Wheat Germ Extract kit (Promega) was used. According to the manufacturer’s instructions, the prepared mixture was incubated at 30°C for 10 min and chilled on ice. Afterwards, 2 µL DPBS buffer or buffer containing different concentrations of OsRIP1 was added to the reaction mixture and incubated for 30 min at 30 °C. After addition of 35 µL nuclease-free water at room temperature, the reaction samples were transferred to a luminometer plate (Greiner Labortechnik, Frickenhausen, Germany) containing 5 µL luciferase assay reagent at 25 °C. The relative luciferase activities of the samples were determined using a microtiter top plate reader (Infinite F200 Pro, Tecan, Mannedorf, Switzerland).
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