In vitro translation was performed in TNT-coupled reticulocyte lysate systems (L4610, Promega) following Promega instruction. Briefly, 1 µg of pcDNA3-DAXX or pcDNA3-p62 was combined with the components as required: TNT reticulocyte lysates, reaction buffer, RNA polymerase, amino acid mixture and RNasin ribonuclease inhibitor. Fifty microlitres of the mixture was incubated at 30 °C for 90 min. In total, 0.5 µl was taken for western blot analysis.
L4610
Manufactured by Promega
The L4610 is a highly sensitive luminometer that measures luminescent signals with high precision. It is designed for a wide range of applications in life science research, including reporter gene assays, ATP measurement, and cell viability studies.
Automatically generated - may contain errors
5 protocols using l4610
1
In vitro Translation of DAXX and p62
2
GST Fusion Protein Pull-down Assay
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GST fusion proteins were expressed in Escherichia coli strain BL21 (Stratagene), purified using standard procedures and stored at −80°C. Proteins were in vitro translated in presence of [35S] methionine (Perkin-Elmer NEG709A500UC) using the reticulocyte lysate-coupled transcription/translation system (Promega L4610) accordingly to manufacturer's instructions. Translation and labeling quality were monitored by SDS-PAGE. GST protein and GST fusion proteins were immobilized on Glutathione Sepharose 4 Fast Flow (GE Healthcare 17-5132-01) and incubated with the in vitro translated proteins in buffer A (40 mM HEPES at pH 7.5, 5 mM MgCl2, 0.2 mM EDTA, 0.5% NP-40, 100 mM KCl) under rotation for 2 h at 4°C. Beads were washed with buffer A, buffer B (40 mM HEPES pH 7.5, 5 mM MgCl2, 0.2 mM EDTA, 0.5% NP-40, 300 mM KCl) and PBS. After washing, beads were boiled in SDS loading buffer and proteins were resolved by SDS-PAGE. Gels were dried and exposed to X-ray films.
3
Purification and Interaction Assay of GST Fusion Proteins
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GST fusion proteins were expressed in Escherichia coli strain BL21 (Stratagene), purified using standard procedures and stored at –80°C. Proteins were in vitro translated in presence of [35S] methionine (Perkin-Elmer NEG709A500UC) using the reticulocyte lysate-coupled transcription/translation system (Promega L4610) accordingly to manufacturer's instructions. Translation and labeling quality were monitored by SDS-PAGE. GST protein and GST fusion proteins were immobilized on Glutathione Sepharose 4 Fast Flow (GE Healthcare 17-5132-01) and incubated together with the in vitro translated proteins in buffer A (40 mM HEPES at pH 7.5, 5 mM MgCl2, 0.2 mM EDTA, 0.5% NP-40, 100 mM KCl) under rotation for 2 h at 4°C. Beads were washed with buffer A, buffer B (40 mM HEPES pH 7.5, 5 mM MgCl2, 0.2 mM EDTA, 0.5% NP-40, 300 mM KCl) and PBS. After washing, beads were resuspended in SDS-PAGE loading buffer and proteins were resolved by SDS-PAGE. Gels were dried and exposed to X-ray films.
4
In Vitro Translation of BC200 RNA
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For in vitro translation, a TnT (transcription/translation)-coupled rabbit reticulocyte lysate system (Promega, L4610) or a standard rabbit reticulocyte lysate system (Promega, L4960) was used. Reaction mixtures (total volume, 10 ml) containing (35S) methionine, buffer, amino acids mixture (minus methionine), increasing amounts of BC200 RNA or its variants, and 0.2 µg luciferase expression plasmid DNA (TnT-coupled rabbit reticulocyte lysate system) or 0.5 µg luciferase mRNA (standard rabbit reticulocyte lysate system) were incubated for 90 min at 30°C as recommended by the manufacturer. Reaction mixtures were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Gels were analyzed using a phospho-image analyzer (FLA-7000; Fuji), and protein bands were quantified using Image J software (NIH).
5
GST-Fusion Protein Purification and Analysis
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GST fusion proteins were expressed in Escherichia coli strain BL21. After purification, the lysates from whole bacterial cells were stored at − 80 °C. Proteins were in vitro translated in presence of [35S] methionine using rabbit reticulocyte lysate system (Promega L4610). GST and GST fusion proteins were immobilized on Glutathione Sepharose beads and incubated with buffer A (40 mM HEPES [pH 7.5], 0.2 mM EDTA, 5 mM MgCl2, 100 mM KCl, 0.5% NP-40) with rotation for 1 h at 4 °C. Beads were washed one time with buffer A, two times with buffer B (40 mM HEPES [pH 7.5], 0.2 mM EDTA, 5 mM MgCl2, 300 mM KCl, 0.5% NP-40) and one time with PBS. Beads were boiled with SDS loading buffer and proteins were separated in SDS-PAGE. Dried gels were exposed to X-ray films (GE Healthcare).
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