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.
Human recombinant ubch5c ube2d3
Manufactured by R&D Systems
Sourced in United States
Human recombinant UbcH5c/UBE2D3 is a laboratory product produced by R&D Systems. It is a recombinant protein that represents the human UbcH5c (also known as UBE2D3) enzyme. UbcH5c is an E2 ubiquitin-conjugating enzyme that plays a role in the ubiquitin-proteasome system.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human ubiquitin, by R&D Systems (5 mentions)
Mops running buffer, by Thermo Fisher Scientific (3 mentions)
Mgatp solution, by R&D Systems (3 mentions)
Recombinant human ubiquitin activating enzyme ube1, by R&D Systems (2 mentions)
Human recombinant gst e1, by R&D Systems (2 mentions)
Rabbit polyclonal anti pparγ antibody, by Cell Signaling Technology (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
5 protocols using human recombinant ubch5c ube2d3
1
In Vitro Ubiquitination Assay
2
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.
3
Ubiquitination and PPAR Regulation Assay
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Human recombinant GST-E1 (50 nM, Boston, Biochem, Cambridge, MA, Cat. #E-306), human recombinant UbcH5c/UBE2D3 (2.5 μM, Boston Biochem, Inc., Cambridge, MA, USA, Cat. #E2-627), human recombinant ubiquitin (250 μM, Boston Biochem, Inc., Cat. #U-100H), human MuRF2 recombinant protein (1 mg, LifeSensors, Cat. #UB305, Malvern, PA, USA), human PPAR-α, -β, and -γ recombinant protein (500 ng, Sigma-Aldrich, Inc., St. Louis, MO, USA, Cat. #SRP2043, Cat. #SRP2044, and Cat. #SRP2045, respectively) were added to reaction buffer (50 mM HEPES, pH 7.5) containing 5 mM MgATP solution (Boston Biochem, Inc., Cat. #B-20) and 0.6 mM DTT then incubated at 37°C for 1 h. The reaction was stopped by adding SDS-PAGE sample buffer and heating, then resolved on a 4–12% Bis–Tris gel with MOPS running buffer (Invitrogen Corp.) and transferred to PVDF membranes for immunoblotting with goat polyclonal anti-MuRF2 antibody (Abcam, Cat. #Ab4387), rabbit polyclonal anti-PPARα antibody (Abcam, Cat. #Ab24509), rabbit polyclonal anti-PPARβ antibody (Millipore, Cat. #AB10094), or rabbit polyclonal anti-PPARγ antibody (Cell Signaling Technology, Cat. #2443).
4
MuRF3 Ubiquitination Assay with PPAR Proteins
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Human recombinant GST-E1 (50 nM, Boston, Biochem, Cambridge, MA, Cat.#E-306), human recombinant UbcH5c/UBE2D3 (2.5 μM, Boston Biochem, Inc., Cambridge, MA, Cat.#E2-627), human recombinant ubiquitin (250 μM, Boston Biochem, Inc., Cat.#U-100H), human MuRF3 recombinant protein (1 mg, LifeSensors, Cat.#UB306, Malvern, PA), human PPAR-α, −β, and -γ recombinant protein (500 ng, Sigma-Aldrich, Inc., St. Louis, MO, Cat.#SRP2043, Cat.#SRP2044, and Cat.#SRP2045, respectively) were added to reaction buffer (50 mM HEPES, pH 7.5) containing 5 mM MgATP solution (Boston Biochem, Inc., Cat.#B-20) and 0.6 mM DTT, then incubated at 37 °C for 1 h. The reaction was stopped by adding SDS-PAGE sample buffer and heating, then resolved on a 4-12 % Bis-Tris gel with MOPS running buffer (Invitrogen Corp.) and transferred to PVDF membranes for immunoblotting with goat polyclonal anti-MuRF3 antibody (Cat.#sc-50252, Santa Cruz Biotechnology), rabbit polyclonal anti-PPARα antibody (Cat.#Ab24509, Abcam), rabbit polyclonal anti-PPARβ antibody (Cat.#AB10094, Millipore), or rabbit polyclonal anti-PPARγ antibody (Cat.#2443, Cell Signaling Technology).
5
Evaluating TRα Ubiquitination by MuRF1
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