An in vitro ubiquitination assay was performed with 20 ng of recombinant human ubiquitin-activating enzyme (UBE1; Boston Biochem), 50 ng of recombinant human UbcH5c/UBE2D3 (Boston Biochem), 2 µg of recombinant human ubiquitin (Boston Biochem), puri ed E3 ubiquitin ligase and substrate in ubiquitylation buffer (40 mM Tris-HCl pH 7.4, 5 mM MgCl 2 , 2 mM ATP, 2 mM DTT). Reactions were carried out in a nal volume of 30 µl for 1 h at 37°C and terminated by the addition of 4× SDS sample loading buffer. The reaction products were analyzed by immunoblotting.
Recombinant human ubiquitin activating enzyme ube1
Manufactured by R&D Systems
Recombinant human ubiquitin-activating enzyme (UBE1) is a laboratory reagent that catalyzes the first step in the ubiquitination pathway, activating ubiquitin prior to its transfer to target proteins. This enzyme is produced in a recombinant system and is suitable for use in biochemical and cell-based assays studying the ubiquitin-proteasome system.
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2 protocols using recombinant human ubiquitin activating enzyme ube1
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In Vitro Ubiquitination Assay
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In Vitro Ubiquitination Assay
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An in vitro ubiquitination assay was performed with 20 ng of recombinant human ubiquitin-activating enzyme (UBE1; Boston Biochem), 50 ng of recombinant human UbcH5c/UBE2D3 (Boston Biochem), 2 µg of recombinant human ubiquitin (Boston Biochem), puri ed E3 ubiquitin ligase and substrate in ubiquitylation buffer (40 mM Tris-HCl pH 7.4, 5 mM MgCl 2 , 2 mM ATP, 2 mM DTT). Reactions were carried out in a nal volume of 30 µl for 1 h at 37°C and terminated by the addition of 4× SDS sample loading buffer. The reaction products were analyzed by immunoblotting.
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