Mgatp solution

Manufactured by R&D Systems

Sourced in United States

MgATP solution is a laboratory reagent containing magnesium adenosine triphosphate (MgATP). It is a cofactor essential for various enzymatic reactions and cellular processes.

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Lab products found in correlation

Recombinant human ubiquitin, by R&D Systems (3 mentions) Mops running buffer, by Thermo Fisher Scientific (3 mentions) Human recombinant ubch5c ube2d3, by R&D Systems (3 mentions) Ube2d1, by R&D Systems (2 mentions) K 982, by R&D Systems (2 mentions) Strep tag 2 antibody hrp conjugate, by Merck Group (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 mgatp solution

Identifying compatible E2 enzymes for BCL6 ubiquitination

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In vitro ubiquitination for identification of compatible E2 conjugating enzymes was performed by following the manufacturer’s instruction (K-982, Boston Biochem), using Strep II-Avi-BCL6⁵⁻³⁶⁰ and Flag-SIAH1^FL. Time-course in vitro ubiquitination was performed by mixing the substrate (BCL6, 2 μM), E3 (SIAH, 0.5 μM), E1 (UBA1, Boston Biochem, 0.2 μM, E2 (UBE2D1, Boston Biochem, 0.5 μM), and ubiquitin (Boston Biochem, 50 μM), with a reaction buffer (B-71, Boston Biochem) containing BI-3802 or DMSO (normalized to 1% DMSO) in 15 μL volume each. Reactions were initiated by adding 5 μL of Mg-ATP solution (B-20, Boston Biochem), incubated for up to 60 min at 37 ºC, and analyzed by western blot using Strep tag II Antibody HRP conjugate (71591–3, Sigma) at 1:4,000.

Słabicki M., Yoon H., Koeppel J., Nitsch L., Roy Burman S.S., Di Genua C., Donovan K.A., Sperling A.S., Hunkeler M., Tsai J.M., Sharma R., Guirguis A., Zou C., Chudasama P., Gasser J.A., Miller P.G., Scholl C., Fröhling S., Nowak R.P., Fischer E.S, & Ebert B.L. (2020). Small molecule-induced polymerization triggers degradation of BCL6. Nature, 588(7836), 164-168.

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Identifying compatible E2 enzymes for BCL6 ubiquitination

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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).

He J., Quintana M.T., Sullivan J., L Parry T., J Grevengoed T., Schisler J.C., Hill J.A., Yates C.C., Mapanga R.F., Essop M.F., Stansfield W.E., Bain J.R., Newgard C.B., Muehlbauer M.J., Han Y., Clarke B.A, & Willis M.S. (2015). MuRF2 regulates PPARγ1 activity to protect against diabetic cardiomyopathy and enhance weight gain induced by a high fat diet. Cardiovascular Diabetology, 14, 97.

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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).

Quintana M.T., He J., Sullivan J., Grevengoed T., Schisler J., Han Y., Hill J.A., Yates C.C., Stansfield W.E., Mapanga R.F., Essop M.F., Muehlbauer M.J., Newgard C.B., Bain J.R, & Willis M.S. (2015). Muscle ring finger-3 protects against diabetic cardiomyopathy induced by a high fat diet. BMC Endocrine Disorders, 15, 36.

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Evaluating TRα Ubiquitination by MuRF1

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To evaluate direct ubiquitination of TRα by MuRF1, human recombinant E1 (50 nM, Cat. #E-305; Boston, Biochem, Cambridge, MA, USA), human recombinant UbcH5c/UBE2D3 (2.5 mM, Cat. #E2-627; Boston Biochem, Inc., Cambridge, MA), human recombinant ubiquitin (250 μM, Cat. #U-100 H; Boston Biochem), human recombinant his-6-MuRF1 protein (1 mg, Cat. #E3-100; Boston Biochem), and human TRα recombinant protein (500 ng; Abnova, Walnut, CA, USA) were added to the reaction buffer (50 mM HEPES, pH 7.5) containing 5 mM MgATP solution (Boston Biochem) and 0.6 mM DTT and were incubated overnight at 30°C. The reaction was stopped by SDS–PAGE sample buffer and heating, then resolved on a 4–12% Bis–Tris gel with MOPS running buffer (Invitrogen), and transferred to PVDF membranes for immunoblotting with rabbit polyclonal anti-TRα antibody (Thermo Scientific Pierce, Inc., Rockford, IL, USA) or rabbit polyclonal anti-MuRF1 antibody (R&D Systems).

Wadosky K.M., Berthiaume J.M., Tang W., Zungu M., Portman M.A., Gerdes A.M, & Willis M.S. (2016). MuRF1 mono-ubiquitinates TRα to inhibit T3-induced cardiac hypertrophy in vivo. Journal of molecular endocrinology, 56(3), 273-290.

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