The TnT Quick Coupled Transcription and Translation system (Promega, Madison, USA) was used to generate [35S]-labeled HBsAg protein from the S gene cloned in pcDNA3.1 vector, using the T7 promoter present on the vector.20 (link) Briefly, rabbit reticulocyte lysate was mixed with 1 mg of plasmid DNA, 2 µL of [35S]-methionine (Amersham, 1,000 Ci/mmol at 10 mCi/mL) and nuclease-free water to a final volume of 50 mL. The reaction mixture was incubated for 90 min at 30 ℃. Expression of each construct was determined by 12 % SDS-PAGE. After fixation for 30 min, the gel was treated with Amplify solution (Amersham, Little Chalfont, UK) and the radio-labeled protein was detected by exposure to X-ray film.
Amplify solution
Manufactured by Cytiva
Sourced in United Kingdom, United States
Amplify solution is a laboratory reagent used to facilitate and enhance nucleic acid amplification processes. It functions as a reaction buffer, providing the necessary components to support and optimize the amplification of DNA or RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
35s methionine, by Cytiva (2 mentions)
Tnt quick coupled transcription and translation system, by Promega (1 mentions)
Tnt coupled reticulocyte lysate system, by Promega (1 mentions)
Xt sample buffer, by Bio-Rad (1 mentions)
Biomax ms film, by Kodak (1 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (1 mentions)
3 protocols using amplify solution
1
Radioactive Protein Expression Using TnT System
2
Radiolabeled Protein Production Workflow
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Radiolabeled DUX1, DUX4, DUX4-t, IPO13 and luciferase (positive control) proteins were produced by transcription/translation (T/T) in vitro (TNT Coupled Reticulocyte Lysate system, Promega) according to the manufacturer’s instructions using pCIneo-DUX1, pCIneo-DUX4, pCRT7/NT-DUX4-t, pGEM7Z-IPO13 or a luciferase vector in the presence of T7 or Sp6 RNA polymerase and 20 μCi L-35S-cysteine (Amersham Biosciences, Roosendaal, The Netherlands). To check the T/T efficiency, the products were boiled for 5 min at 95°C in XT sample buffer (Bio-Rad, Hercules, CA) or SDS loading buffer in the presence of a reducing agent (Fermentas, St. Leon-Rot Germany) and analyzed by SDS-PAGE. The gel was incubated for 30 min in the Amplify solution (Amersham Biosciences), air dried and subjected to autoradiography.
3
Fusion Protein Expression via TNT
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For the generation of fusion proteins, the TNT T7 Coupled Reticulocyte Lysate System (Promega, Mannheim, Germany) was used together with 35S‐methionine (Amersham, Freiburg, Germany) following the manufacturer's protocol. For this purpose, 1 μg of each construct and 0.5 μg of control plasmid MKKS‐pGBKT7 (encoding a C‐myc‐tagged MKKS protein of 62 kD) were used. After completed TNT reaction, 20 μL of 6 × SDS‐PAGE sample buffer was added to 5 μL of the samples, boiled for 5 min and separated by SDS‐PAGE on a 12% acrylamid gel. The gel was treated with fixation buffer and Amplify Solution (Amersham) and exposed overnight at room temperature using Kodak BioMax MS film (Kodak, Rochester, NY, USA).
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