HOIL-1L-6xHIS and HOIP-K1056R-6xHIS were cloned into the pET-duet-1 vector, expressed in BL21(DE3) RIPL bacteria (Agilent Technologies), and purified from cultures as previously described (5 (link)). HOIP-Cterm (amino acids 699 to 1072), E1 (Ube1), E2 (UbcH5c), and ubiquitin were purchased from Boston Biochem. In vitro linear-ubiquitination assay mixtures contained 200 ng E1, 400 ng E2, 1 µg HOIL-1L/HOIP-6xHIS purification prep, 20 mM Tris (pH 7.5), 5 mM dithiothreitol (DTT), 5 mM MgCl2, 2 mM Mg-ATP (Boston Biochem), and 10 µg ubiquitin. Assay mixtures were incubated for various times at 37°C, and reactions were stopped by adding LDS (lithium dodecyl sulfate) sample buffer (Life Technologies) and heating at 70°C for 10 min.
Protocol detail
Purification and In Vitro Ubiquitination
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Amino acids
Assay
Bacteria
Buffer
Dithiothreitol
Lithium dodecyl sulfate
Mgcl2
Tris
Ube1
Ubiquitin
Ubiquitination
Vector
Corresponding Organization :
Other organizations : Keck Graduate Institute, Abbott Fund, University of Iowa, Kyoto University, University of Southern California
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Variable analysis
independent variables
- Expression of HOIL-1L-6xHIS and HOIP-K1056R-6xHIS in BL21(DE3) RIPL bacteria
- Incubation time of the in vitro linear-ubiquitination assay mixtures
- Linear ubiquitination activity of the HOIL-1L/HOIP-6xHIS purification preparation
- Concentration of E1 (Ube1)
- Concentration of E2 (UbcH5c)
- Concentration of HOIL-1L/HOIP-6xHIS purification preparation
- PH of the reaction buffer (20 mM Tris, pH 7.5)
- Concentration of DTT (5 mM)
- Concentration of MgCl2 (5 mM)
- Concentration of Mg-ATP (2 mM)
- Concentration of ubiquitin (10 μg)
- Positive control: Not specified
- Negative control: Not specified
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