Tnt t7 quick coupled transcription translation system kit

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).

Protein synthesis inhibition activity for non-reduced or reduced S. nigra RIPs (SNA-I, SNA-V or SNLRP) was determined using the TnT T7 Quick Coupled Transcription/Translation System Kit (Promega, Mannheim, Germany) based on a cell-free system [20 (link)]. The lectins SNA-II and SNA-IV were also included in this assay. According to manufacturer’s instructions, the prepared mixture was incubated at 30°C for 10 min and chilled on ice. Afterwards, 2 μl PBS or PBS containing different concentrations of S. nigra RIPs or lectins were 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 at 562 nm for 10 sec using a microtiter top plate reader (Infinite 200, Tecan, Mannedorf, Switzerland) with an initial delay of 2 sec.

Glutathione‐S‐transferase (GST) pull‐down assay was used to verify the interaction between AFP and RAR. A GST‐RAR clone was purchased from Beijing FunGenome. GST pull‐down experiments were performed using the TNT T7 Quick Coupled Transcription/Translation System Kit (Promega), followed by Western blotting to evaluate the interaction between TNT system translation products and the GST‐RAR fusion proteins.