Purified His-HA-TMEPAI or His-HA-TMEPAI-2YA/KR proteins were incubated with recombinant E1 (Boston Biochem), recombinant UbcH5C (Boston Biochem), GST-Nedd4 and methylated ubiquitin or His-tagged ubiquitin (Boston Biochem) in reaction buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.3 mM DTT, 2 mM ATP) for 60 min at 30 °C. The reaction products were analyzed by SDS-PAGE and Western blot.
Recombinant e1
Manufactured by R&D Systems
Sourced in United States
Recombinant E1 is a protein produced using recombinant DNA technology. It is a component of the hepatitis C virus.
Automatically generated - may contain errors
Lab products found in correlation
Ubiquitin, by R&D Systems (4 mentions)
Recombinant ubch5c, by R&D Systems (3 mentions)
Ubiquitin reaction buffer, by R&D Systems (2 mentions)
Energy restoration system, by R&D Systems (2 mentions)
Ubch5c, by R&D Systems (2 mentions)
Coomassie brilliant blue, by Bio-Rad (2 mentions)
Sypro ruby protein gel stain, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti ha antibody, by Roche (1 mentions)
Anti ha affinity gel, by Merck Group (1 mentions)
Ubc13 uev1a, by R&D Systems (1 mentions)
7 protocols using recombinant e1
1
In Vitro Ubiquitination Assay
2
Deubiquitination and Self-Ubiquitination Assays
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Deubiquitination activity of CYLD-CP5 was evaluated as below. One hundred nM of purified CYLD-CP5 or Venus-CP5 was mixed with 1 μM recombinant M1-linked tetra ubiquitin (R&D Systems) in 12 μL of reaction mixture containing 50 mM Tris-HCl (pH 7.5), 5 mM DTT. The reaction mixtures were incubated at 30°C for 3 hours, then the reaction was terminated by boiling in SDS sample buffer. Degraded ubiquitin chain was visualized by SDS-PAGE and SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific).
In vitro self-ubiquitination assay of MARCH3 was conducted as follows. Five μL of purified MARCH3-CP5 or Venus-CP5 was mixed with 4 μM recombinant HA-ubiquitin (Boston Biochem) in 20 mM Tris-HCl (pH 7.5), 200 μM DTT, 5 mM MgCl2, 3 mM ATP, 40 nM recombinant E1 (Boston Biochem) and 300 nM recombinant UbcH6 (Enzo Life Sciences). The reaction mixture was incubated at 30°C for 3 hours. The reaction mixture was boiled in SDS sample buffer and subjected to SDS-PAGE followed by immunoblot analysis using HRP conjugated anti-HA antibody (Roche Life Science).
In vitro self-ubiquitination assay of MARCH3 was conducted as follows. Five μL of purified MARCH3-CP5 or Venus-CP5 was mixed with 4 μM recombinant HA-ubiquitin (Boston Biochem) in 20 mM Tris-HCl (pH 7.5), 200 μM DTT, 5 mM MgCl2, 3 mM ATP, 40 nM recombinant E1 (Boston Biochem) and 300 nM recombinant UbcH6 (Enzo Life Sciences). The reaction mixture was incubated at 30°C for 3 hours. The reaction mixture was boiled in SDS sample buffer and subjected to SDS-PAGE followed by immunoblot analysis using HRP conjugated anti-HA antibody (Roche Life Science).
3
Ubiquitination Assay for c-Maf, HERC4, and USP5
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This protocol was adapted from a previous report.22 (link) In brief, HA-c-Maf, HERC4, and Flag-USP5 plasmids were transfected into HEK293T cells, respectively. Forty-eight hours later, cells were treated with MG132 for 2 h, followed by cell lysate preparation. To enrich and purify c-Maf, HERC4, and USP5 proteins, individual cell lysates were subjected to IP with HA- (for c-Maf) or Flag- (for HERC4 and USP5) antibody-conjugating agarose beads, respectively, at 4 °C for 12 h. After that, the beads were washed four times with an IP lysis buffer, twice with 1 × ubiquitin reaction buffer (Boston Biochem, Boston, MA, USA) and then resuspended in 20 μl of 1 × ubiquitin reaction buffer containing 200 ng of recombinant E1, 250 ng of recombinant UbcH5c, 10 μg of ubiquitin, 0.5 mM ATP, and 1 × Energy Restoration System (Boston Biochem). The reaction was carried out at 30 °C for 2 h and then terminated by boiling in 2 × SDS loading buffer. ubiquitinated products were resolved by SDS-PAGE and detected by IB analysis.
4
In Vitro Ubiquitination Assay
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This assay was adapted from a previous report [16 (link)]. Briefly, HA-c-Maf and Flag-UBE2O plasmids were transfected into HEK293T cells, respectively. Forty-eight hours later, cells were treated with MG132 for 2 h, followed by cell lysate preparation. To enrich and purify c-Maf and UBE2O proteins, individual cell lysates were subjected to immunoprecipitation with HA- (for c-Maf) or Flag- (for UBE2O) antibody-conjugating agarose beads, respectively, at 4 °C for overnight. After that, the beads were washed four times with an immunoprecipitation lysis buffer, twice with 1× ubiquitin reaction buffer (Boston Biochem, Boston, MA) and then re-suspended in 20 μl of 1× ubiquitin reaction buffer containing 200 ng of recombinant E1, 250 ng of recombinant UbcH5c, 10 μg of ubiquitin, 0.5 mM ATP, and 1× Energy Restoration System (Boston Biochem, Boston, MA). The reaction was carried out at 30 °C for 2 h and then terminated by boiling in the 2× SDS loading buffer. ubiquitinated products were resolved by SDS-PAGE and detected by immunoblotting analysis.
5
Ubiquitination Assay of AMOTL2
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A mixture containing 50 nM E1, 50 nM UbcH5c, purified GST-WWP1, and 1 U of WT or 2KR mutant AMOTL2 were incubated with 200 mM WT or K0 ubiquitin at 37°C in a reaction buffer containing 50 mM Tris-Cl (pH 7.5), 2.5 mM MgCl2, 2 mM DTT, and 2 mM ATP. Samples were then subjected to SDS–PAGE, and resolved proteins were visualized by Coomassie Brilliant Blue (Bio-Rad) staining and Western blot analysis. Recombinant E1, UbcH5c, and ubiquitin were purchased from Boston Biochem.
6
PARP1 Ubiquitination by RNF168 Assay
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To measure the ubiquitination activity of PARP1 by RNF168, 50 nM E1, 50 nM UbcH5c, E3 (WT or mutants of GST-RNF168), and 1 unit of PARP1 were incubated with 200 mM ubiquitin at 37°C in reaction buffer containing 50 mM Tris-Cl (pH 7.5), 2.5 mM MgCl2, 2 mM DTT, and 2 mM ATP. ubiquitinated proteins were detected by immunoblot with anti-ubiquitin antibody. All proteins were separately visualized by Coomassie Brilliant Blue (Bio-Rad). Recombinant E1, UbcH5c and ubiquitin were purchased from Boston Biochem.
7
In Vitro Ubiquitination of VDAC1
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In vitro ubiquitination assays were performed as previously described [31, 32] . An expression vector encoding HA-VDAC1 was transfected into HEK293T cells by FugeneHD reagent. Thirty-six hours after transfection, the cells were collected and lysed in a lysis buffer. HA-VDAC1 was immunoprecipitated with anti-HA-affinity gel (Sigma), and the immunocomplexes were washed five times with the lysis buffer.
HA-beads were then incubated at 30°C for 2 h with gentle agitation in the presence of the following components: HEPES-NaOH (pH 7.9), 5 mM MgCl 2 , 60 mM potassium acetate, 1 mM ATP, 2 mM DTT, 0.5 mM EDTA, 100 ng of recombinant E1 (Boston Biochem, Cambridge, MA), 500 ng of recombinant E2 (UbcH1, UbcH2, UbcH3, UbcH5A, UbcH5B, UbcH5C, UbcH6, UbcH7, UbcH8, Ubc2A, Ubc2B, Ubc13/Uev1a or UbcH2S: Boston Biochem) and 5 µg of His-ubiquitin with or without recombinant FLAG-RNF207(WT). The reaction was terminated by the addition of an SDS sample buffer containing 4% β-mercaptoethanol and heating at 95°C for 5 min. Samples were subjected to Western blot analysis with anti-HA antibody.
HA-beads were then incubated at 30°C for 2 h with gentle agitation in the presence of the following components: HEPES-NaOH (pH 7.9), 5 mM MgCl 2 , 60 mM potassium acetate, 1 mM ATP, 2 mM DTT, 0.5 mM EDTA, 100 ng of recombinant E1 (Boston Biochem, Cambridge, MA), 500 ng of recombinant E2 (UbcH1, UbcH2, UbcH3, UbcH5A, UbcH5B, UbcH5C, UbcH6, UbcH7, UbcH8, Ubc2A, Ubc2B, Ubc13/Uev1a or UbcH2S: Boston Biochem) and 5 µg of His-ubiquitin with or without recombinant FLAG-RNF207(WT). The reaction was terminated by the addition of an SDS sample buffer containing 4% β-mercaptoethanol and heating at 95°C for 5 min. Samples were subjected to Western blot analysis with anti-HA antibody.
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