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.
Neg709a500uc
Manufactured by PerkinElmer
The NEG709A500UC is a lab equipment product from PerkinElmer. It is designed to perform a core function within the laboratory setting. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4 fast flow, by GE Healthcare (2 mentions)
L4610, by Promega (2 mentions)
Tnt quick coupled translation system, by Promega (1 mentions)
Trypsin, by Merck Group (1 mentions)
Ribomax large scale rna production kit, by Promega (1 mentions)
L4380, by Promega (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
6 protocols using neg709a500uc
1
GST Fusion Protein Pull-down Assay
2
In Vitro Mitochondrial TFEB Import
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Purified mitochondria were resuspended in the mitochondria isolation buffer (MIB, 220 mM mannitol, 140 mM Sucrose, 10 mM HEPES/KOH pH 7.4, 1 mM EGTA/KOH).
For in vitro import assay WT and MLS-TFEB cDNA were cloned into the pGEM-4 vector and transcribed in vitro using the TNT Quick coupled translation system (L1170, Promega), by incubating 200 ng of DNA with 7 μL of TNT-T7 reticulocyte lysate mix and 0.5 µL of 35S-labeled methionine (NEG709A500UC, PerkinElmer) for 1 h at 30 °C at 600 rpm. Next, the mitochondrial pellets were resuspended into mitochondrial import buffer (500 mM Sucrose, 10 mM MgAc, 160 mM KAc, 20 mM sodium succinate and 40 mM Hepes-KOH pH 7.4, 0.3 M ATP, and 1 M fresh DTT). Then, mitochondria were incubated with the lysate containing TFEB transcribed in vitro and the membrane potential was blocked by adding 1 µL of 20 mM CCCP at different time points. Mitochondria were also treated with Trypsin (T1426, Sigma Aldrich) (25 μg/mL for 10 min) followed by Trypsin inhibitor soya bean (Merck, 10109886001) (250 μg/mL for 15 min). 1% Triton X-100 was eventually added to permeabilize the mitochondrial membranes and allow the Trypsin to reach the mitochondrial matrix. The mitochondrial pellet was lysed in denaturing conditions and proteins were detected by autoradiography.
For in vitro import assay WT and MLS-TFEB cDNA were cloned into the pGEM-4 vector and transcribed in vitro using the TNT Quick coupled translation system (L1170, Promega), by incubating 200 ng of DNA with 7 μL of TNT-T7 reticulocyte lysate mix and 0.5 µL of 35S-labeled methionine (NEG709A500UC, PerkinElmer) for 1 h at 30 °C at 600 rpm. Next, the mitochondrial pellets were resuspended into mitochondrial import buffer (500 mM Sucrose, 10 mM MgAc, 160 mM KAc, 20 mM sodium succinate and 40 mM Hepes-KOH pH 7.4, 0.3 M ATP, and 1 M fresh DTT). Then, mitochondria were incubated with the lysate containing TFEB transcribed in vitro and the membrane potential was blocked by adding 1 µL of 20 mM CCCP at different time points. Mitochondria were also treated with Trypsin (T1426, Sigma Aldrich) (25 μg/mL for 10 min) followed by Trypsin inhibitor soya bean (Merck, 10109886001) (250 μg/mL for 15 min). 1% Triton X-100 was eventually added to permeabilize the mitochondrial membranes and allow the Trypsin to reach the mitochondrial matrix. The mitochondrial pellet was lysed in denaturing conditions and proteins were detected by autoradiography.
3
Synthetic protein translation assay
4
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.
5
Reovirus σNS Protein Characterization
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Reovirus σNS proteins were expressed from plasmids in vitro using the TNT T7 polymerase-coupled rabbit-reticulocyte lysate system (Promega, L4610) (19 (link)). Reactions were supplemented with [35S]methionine (Perkin Elmer, NEG709A500UC), incubated at 30°C for 1.5 h, and terminated with a 4-fold dilution in stop buffer (20 mM HEPES-KOH [pH 7.4], 100 mM potassium acetate, 5 mM magnesium acetate, 5 mM EDTA, 2 mM methionine, and freshly supplemented to contain 1 mM dithiothreitol [DTT] and 2 mM puromycin). Terminated reactions were used for proteolysis and RNA-dependent oligomerization assays.
Proteolysis assays were conducted by incubating translation reactions with 1 μg/ml proteinase K (Sigma) at 37°C for 0, 5, 10, or 15 min. Samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
RNA-dependent oligomerization assays were conducted by incubating translation reactions with 10 μg of RNase A (Thermo Fisher) or 125 mM NaCl at 37°C for 1 h. Samples were prepared for native PAGE and SDS-PAGE.
Proteolysis assays were conducted by incubating translation reactions with 1 μg/ml proteinase K (Sigma) at 37°C for 0, 5, 10, or 15 min. Samples were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
RNA-dependent oligomerization assays were conducted by incubating translation reactions with 10 μg of RNase A (Thermo Fisher) or 125 mM NaCl at 37°C for 1 h. Samples were prepared for native PAGE and SDS-PAGE.
6
In Vitro Protein Translation Assay
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