Ubiquitin variants were concentrated to 4 mM. K48-linked diubiquitin was synthesized in 1 ml of 50 mM Tris–HCl, pH 8.0, 2 mM TCEP supplemented with 1× energy regeneration solution (BostonBiochem), 100 nM His6-Ube1 (BostonBiochem), 2.5 μM E2-25K (BostonBiochem), 1 mM ubiquitinG76C, and 1 mM ubiquitinK48R. K63-linked diubiquitin was synthesized in the same buffer supplemented with 1× energy regeneration solution, 100 nM His6-Ube1, 2.5 μM His6-UBE2N/Uev1a complex (BostonBiochem), 1 mM ubiquitinG76C, and 1 mM ubiquitinK63R. Reactions were incubated at 30°C for 16 h then flowed through a 1 ml Ni2+-NTA superflow cartridge (QIAGEN) to extract the His6-tagged E1/E2 enzymes. Unreacted ubiquitin and diubiquitin were then separated on a HiLoad 26/60 Superdex 75 pg (GE Healthcare) equilibrated in 20 mM Hepes, pH 7.5, 150 mM NaCl, and 2 mM TCEP.
Energy regeneration solution
Manufactured by R&D Systems
Sourced in United States
The Energy Regeneration Solution is a lab equipment product designed to efficiently capture and store energy generated during experiments or testing procedures. Its core function is to enable the recycling and reuse of energy, thereby reducing overall energy consumption and costs associated with laboratory operations.
Automatically generated - may contain errors
Lab products found in correlation
Ha ubiquitin, by R&D Systems (2 mentions)
Ubiquitin, by R&D Systems (2 mentions)
His6 ube1, by R&D Systems (2 mentions)
Ni2 nta superflow cartridge, by Qiagen (2 mentions)
Hiload 26 60 superdex 75 pg, by GE Healthcare (2 mentions)
Ubiquitin e1, by R&D Systems (1 mentions)
Ube2h5b, by R&D Systems (1 mentions)
1 step human coupled ivt kit dna, by Thermo Fisher Scientific (1 mentions)
Amp glo assay, by Promega (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Adenosine triphosphate (atp), by Sino Biological (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Ubch5b, by R&D Systems (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Chir99021, by Merck Group (1 mentions)
Ubch5a, by R&D Systems (1 mentions)
Pd0325901, by Merck Group (1 mentions)
Ssea 1, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibodies against mouse and rabbit igg, by Promega (1 mentions)
6 protocols using energy regeneration solution
1
Synthesis of Ubiquitin Linkage Types
2
In vitro Ubiquitination Assay Protocol
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In vitro ubiquitination assays were performed as described previously (20 (link)). Ubiquitin E1 (UBE1), ubiquitin E2 (UbcH5a), and HA-ubiquitin were obtained from a commercial source (Boston Biochem). FLAG-ZIC2 was used as a substrate. Ubiquitin conjugation reactions were performed using a final protein concentration of E1 of 60 nM, E2 of 300 nM, E3 (K-Rta) of 100 nM, and ubiquitin of 10 μM in ubiquitin conjugation reaction buffer supplemented with an energy regeneration solution (Boston Biochem) for 2.5 h at 37°C.
3
Phosphorylation and Ubiquitination of cCBL
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For cCBL phosphorylation, 50 ng of Y505F-Lck [18 (link)] were mixed with the cCBL IP (1 mg cell extract) or r-(recombinant) cCBL (200 ng) and incubated in Lck kinase buffer (25 mM Tris-HCl pH 7.4, 5 mM MnCl2, 50 μM ATP) for 15 min at 25 °C. cCBL reactions were performed in ubiquitination buffer (40 mM Tris-HCl pH 7.4, 2 mM DTT, 5 mM MgCl2, 5mM ATP). The reaction volumes (40 μL) contained 200 ng purified recombinant (r)-cCBL (Merck-Millipore, Burlington, MA, USA), 5 μg ubiquitin, 250 ng UBA1, and 250 ng of UBE2H5B or UBE2N/Ubc13 (all from Boston Biochem, Cambridge, MA, USA). Parallel reactions with cCBL and WWP1 (20 μL) were performed in ubiquitination buffer using r-cCBL (200 ng) or r-WWP1 (Ubiquigent, Dundee, Scotland, UK) (200 ng), UBE2H5B (300 ng), UBA1 (50 ng), and energy regeneration solution (Boston Biochem). To test for PTEN ubiquitination, GST-PTEN (100–400 ng, indicated) was purified by pull-down, suspended in ubiquitination buffer, and added to the reaction. Reactions (2 h, 30 °C) were terminated with 1× Laemli buffer.
4
In vitro Ubiquitination Assay for UBE2A
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In vitro translation of UBE2A proteins was performed with 1-Step Human Coupled IVT Kit-DNA (Thermo-Fischer-Scientific) following the manufacturer’s instructions. For ubiquitination assay we incubated 15 μg of UBE2A proteins with 1 μg of GST-Ubiquitin (Enzo-Life-Sciences, NY, USA), 0.2 ng of ubiquitin activating enzyme (E1) (Enzo-Life-Sciences), 2 mM ATP, energy regeneration solution (BostonBiochem, MA, USA), 2 mM MgCl2, 2 mM KCl, 16 μg of BA/F3_BCR-ABL1 whole cell lysate in 50 mM TrisHCl (ph7.5). The reactions were incubated for 20 minutes at 37°C. The products were analyzed by western blotting.
For the enzymatic activity of WT and mutated UBE2A, 15 μg of UBE2A in vitro synthesized protein were used. The AMP-Glo Assay (Promega catalog v5011) was used in order to quantify the amount of AMP generated by the ubiquitin conjugation machinery, composed of 170 ng/μL ubiquitin protein, 15 ng/μL UBA1 and 50 μM ATP (SignalChem).
The production of AMP from ATP is directly proportional to the enzymatic activity of the ubiquitination machinery and therefore it was used to measure the ubiquitination in the presence of WT and mutated UBE2A. The AMP signal was detected using the AMP detection solution (Promega) and a TECAN reading plate (Infinite F200Pro TECAN).
For the enzymatic activity of WT and mutated UBE2A, 15 μg of UBE2A in vitro synthesized protein were used. The AMP-Glo Assay (Promega catalog v5011) was used in order to quantify the amount of AMP generated by the ubiquitin conjugation machinery, composed of 170 ng/μL ubiquitin protein, 15 ng/μL UBA1 and 50 μM ATP (SignalChem).
The production of AMP from ATP is directly proportional to the enzymatic activity of the ubiquitination machinery and therefore it was used to measure the ubiquitination in the presence of WT and mutated UBE2A. The AMP signal was detected using the AMP detection solution (Promega) and a TECAN reading plate (Infinite F200Pro TECAN).
5
Ubiquitination Assay for Rmnd5 and HDM2
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A 25 μl ubiquitination reaction contained 0.25 μg of E1 (yeast), 0.6 μg of UbcH5b, 10 μg of HA-ubiquitin, 1 μl of energy regeneration solution (all BostonBiochem, Cambridge), 2 μl ATP (100 mM, pH7.5), 2.5 μl ubiquitin reaction buffer (500 mM Tris-HCl, pH7.5, 500 mM NaCl, 100 mM MgCl2, 10 mM DTT, and 250 μM ZnCl2) and 2.25 μg of purified Rmnd5 or HDM2 (Enzo Lifescience) as a positive control. The reactions were incubated at 30°C for 3 h. Ubiquitination of protein was monitored by Western blot analysis with polyclonal anti-HA antibody (Sigma).
6
Ubiquitination Assay Protocol
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The following reagents used in this study were purchased from the indicated sources: CHIR99021 (Millipore, 3 μM), PD0325901 (Sigma, 1 μM), H-89 (Beyotime, 10 μM), antibodies against GAPDH (Santa Cruz, 1:1,000), Actin (Cell Signaling, 1:1,000), green fluorescent protein (GFP; MBL 1:4,000), Flag (Sigma 1:4,000), march5 (Abcam, 1:500), Prkar1a (Cell Signaling, 1:1,000), ERK (Cell Signaling, 1:1,000), p-ERK (Thr202/Tyr204) (Cell Signaling, 1:1,000), phosphorylated Raf (Ser259) (Santa Cruz, 1:1,000), Oct4 (Santa Cruz, 1:1,000) and Sox2 (Millipore, 1:1,000), SSEA1 (Santa Cruz, 1:50), HRP-conjugated secondary antibodies against mouse and rabbit IgG (Promega). Ubiquitin, E1, UbcH5a and 1 × energy regeneration solution were purchased from Boston Biochem (USA).
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