Purified proteins were obtained by expressing the proteins in an Escherichia coli strain BL21 or commercially. Bacteria were grown in LB medium, induced with 0.25mM isopropyl-β-D-thiogalactoside (IPTG) for 2 hours at 37°C, and cultured at 16°C for 12 hours. The cells were then lysed using appropriate lysis buffer supplemented with Protease Inhibitor Cocktail (Roche). The proteins were purified by binding to appropriate beads for 3 hours at 4°C. The beads were washed with lysis buffer twice and the protein was subsequently eluted. Recombinant human UBA1, UBE2D3/UBCH5C, UBE2N/UBE2V2 complex, UBE2N (UBC13)/UEV1A complex, USP2, RNF8, RNF168, L3MBTL2, and ubiquitin (wild-type and mutants) used for in vitro ubiquitylation assays were purchased from Boston Biochem, Biotechne, Abnova and Origene.
Rnf168
Manufactured by R&D Systems
RNF168 is a recombinant human protein that functions as an E3 ubiquitin-protein ligase. It is involved in the DNA damage response pathway.
Automatically generated - may contain errors
Lab products found in correlation
Ubiquitin, by R&D Systems (3 mentions)
Recombinant human uba1, by R&D Systems (2 mentions)
Ube2d3 ubch5c, by R&D Systems (2 mentions)
Ube2n ube2v2 complex, by R&D Systems (2 mentions)
Ube2n ubc13 uev1a complex, by R&D Systems (2 mentions)
L3mbtl2, by R&D Systems (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ubiquitin activating enzyme e1, by R&D Systems (1 mentions)
3 protocols using rnf168
1
Purification of Recombinant Ubiquitylation Enzymes
2
Ubiquitin Modification of Nucleosomes
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Assays were performed essentially as described [10] (link). Briefly, 2.5 µg of recombinant mononucleosomes were incubated in a 50 µl reaction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 µM ZnOAc, 1 mM DTT, 30 nM ubiquitin activating enzyme E1 (Boston Biochem), 1.5 µM ubiquitin conjugating enzyme UbcH5a (Boston Biochem), 4 µM RNF168 (1–113) or RING1B/BMI1 complex, 22 µM ubiquitin (Boston Biochem) and 3.33 mM ATP at 30°C for 4 h. The reaction was terminated by addition of SDS-PAGE loading buffer. Assays with free histones were carried out with 10 µM of H2A or H2AX and the reactions were incubated overnight at 30 C. The samples were boiled and loaded on 15% SDS-PAGE gels, transferred to a nitrocellulose membrane, probed using specific antibodies and detected as described above.
3
Purification of Recombinant Ubiquitylation Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified proteins were obtained by expressing the proteins in an Escherichia coli strain BL21 or commercially. Bacteria were grown in LB medium, induced with 0.25mM isopropyl-β-D-thiogalactoside (IPTG) for 2 hours at 37°C, and cultured at 16°C for 12 hours. The cells were then lysed using appropriate lysis buffer supplemented with Protease Inhibitor Cocktail (Roche). The proteins were purified by binding to appropriate beads for 3 hours at 4°C. The beads were washed with lysis buffer twice and the protein was subsequently eluted. Recombinant human UBA1, UBE2D3/UBCH5C, UBE2N/UBE2V2 complex, UBE2N (UBC13)/UEV1A complex, USP2, RNF8, RNF168, L3MBTL2, and ubiquitin (wild-type and mutants) used for in vitro ubiquitylation assays were purchased from Boston Biochem, Biotechne, Abnova and Origene.
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