Ubch5b e2

Manufactured by R&D Systems

Sourced in United States

UbcH5b (E2) is a recombinant human Ubiquitin-conjugating enzyme E2 B (UBE2B) protein. UBE2B is an E2 enzyme that functions in the ubiquitin-proteasome pathway by catalyzing the covalent attachment of ubiquitin to target proteins.

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Lab products found in correlation

Ubiquitin, by R&D Systems (6 mentions) E1 ube1, by R&D Systems (5 mentions) Ha ubiquitin, by R&D Systems (2 mentions) Inorganic pyrophosphatase, by Merck Group (2 mentions) K48r his6 tagged, by R&D Systems (2 mentions) Ni nta agarose bead, by Qiagen (2 mentions) Ha tagged ubiquitin ha ub, by R&D Systems (2 mentions) Adenosine triphosphate (atp), by Merck Group (2 mentions) Flag ubiquitin, by R&D Systems (2 mentions) Ubiquitin aldehyde, by R&D Systems (1 mentions) Glutathione agarose beads, by Thermo Fisher Scientific (1 mentions) Mg132, by R&D Systems (1 mentions) Anti ha antibody, by Merck Group (1 mentions) Anti gst antibody, by Merck Group (1 mentions) Tricine sample buffer, by Bio-Rad (1 mentions) Typhoon imager, by GE Healthcare (1 mentions) Anti ubiquitin, by Santa Cruz Biotechnology (1 mentions) Anti ha, by Merck Group (1 mentions) Anti ubiquitin, by Merck Group (1 mentions) Creatine phosphokinase, by Merck Group (1 mentions)

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UbcH5b (22 citations)

16 protocols using ubch5b e2

In vitro Nedd4-Pax7 Ubiquitination Assay

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1μg of GST-Nedd4 or GST-only were pre-bound to 50μl glutathione-agarose beads (Thermo Scientific, USA) for 2 h at 4°C in pull-down buffer. Immobilized GST-proteins were incubated with 5μg of purified Pax7 protein in a 100μl reaction containing: 10μg of recombinant HA-Ubiquitin, 200ng of Ubiquitin Activating Enzyme UBE1 (E1), 300ng of UbcH5b (E2), 1X Energy regeneration solution (ERS) (Boston Biochem, USA), and 50mM HEPES, pH 7.5 buffer. After 1h incubation at 30 °C, supernatants were recovered for further analysis. Presence of Nedd4 in gluthatione-agarose beads was confirmed by Western blotting (Fig. S3B). E3 activity was determined by detection of auto-ubiquitinated Nedd4 in parallel assays (data not shown), as described [57 (link)].
Alternatively 5μg of GST-Pax7 or GST-only proteins were incubated in a 50μl reaction with S-100 HeLa cell fraction 3.7mg/ml as UPS source, supplemented with 5μg of HA-Ubiquitin, 1X ERS and 8μM MG132 and 250ng/μl ubiquitin aldehyde (Boston Biochem, USA). Reactions were incubated at 30°C for 1 h and diluted in 500μl of GST pull-down buffer prior to analysis.

Bustos F., de la Vega E., Cabezas F., Thompson J., Cornelison D., Olwin B.B., Yates JR I.I.I, & Olguín H.C. (2015). NEDD4 REGULATES PAX7 LEVELS PROMOTING ACTIVATION OF THE DIFFERENTIATION PROGRAM IN SKELETAL MUSCLE PRECURSORS. Stem cells (Dayton, Ohio), 33(10), 3138-3151.

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IpaH Ubiquitination Assay Protocol

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Shigella flexneri genes encoding IpaH proteins were cloned into pGEX4T-1, expressed in E. coli BL21 and purified using glutathione coated beads. His₆-UbcH5b and His₆-UbE1 were expressed in E. coli BL21 and purified using Ni-NTA agarose beads (Qiagen). LUBAC (Flag-HOIL-1L/Myc-HOIP/Flag-His₆-SHARPIN) was expressed in HeLa cells and purified using Ni-NTA agarose beads (Qiagen). LUBAC purified from HeLa cells was then used in an Ub reaction, containing 0.1 μM UbE1 (E1), 2.5 μM UbcH5b (E2), 5 μM (Flag, HA or His₆ tagged) Ub (BostonBiochem), 0.5 mM MgSO₄, 0.5 mM ATP, 20 units/ml inorganic pyrophosphatase (Sigma Aldrich), 1 mM DTT and 50 nM GST-IpaH (E3). After 0, 20, 60 or 90 minutes incubation at 37°C samples were electrophoresed on an SDS PAGE buffer and a Western blot was stained with anti-Myc antibodies. Wild type Ub (Flag, HA, His₆ tagged), K48 only Ub (His₆ tagged) and K48R (His₆ tagged) were purchased from BostonBiochem.

de Jong M.F., Liu Z., Chen D, & Alto N.M. (2016). Shigella flexneri suppresses NF-kB activation by inhibiting linear ubiquitin chain ligation. Nature microbiology, 1(7), 16084.

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RipAW Ubiquitination Assay Protocol

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R. solanacearum GMI1000 RipAW was cloned into pGEX6p‐1 vector with BamHI/XhoI. PCR‐based site‐directed mutagenesis was used to mutate the cysteine 177 of RipAW into an alanine or a serine. The obtained pGEX‐RipAW, pGEX‐RipAW (C177A) and pGEX‐RipAW (C177S) were transformed into E. coli BL21. The GST‐tagged RipAW, RipAW (C177A), and RipAW (C177S) were purified from the IPTG‐induced cell cultures by glutathione resin beads (Sigma) and detected by western blot with anti‐GST antibody (Sigma).
Two micrograms of purified GST‐tagged RipAW and its derivatives were incubated with 0.5 μg human E1, 2 µg UbcH5B E2, and 2 µg HA‐ubiquitin (Boston Biochem) in 40 µl of reaction buffer (25 mM Tris.HCl pH 7.5, 50 mM NaCl, 5 mM ATP, 10 mM MgCl₂, 0.1 mM dithiothreitol) for 2 h at 37°C. The reaction was stopped by addition of 5× SDS‐loading buffer. The reaction mixtures were separated by SDS‐PAGE and detected with anti‐GST antibody and anti‐HA antibody (Sigma).

Niu Y., Fu S., Chen G., Wang H., Wang Y., Hu J., Jin X., Zhang M., Lu M., He Y., Wang D., Chen Y., Zhang Y., Coll N.S., Valls M., Zhao C., Chen Q, & Lu H. (2021). Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses. Molecular Plant Pathology, 23(2), 188-203.

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In vitro Ubiquitination Assay for p53

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p53-based peptide ubiquitination was evaluated using an in vitro ubiquitination assay. The reaction consisted of 10 mM Tris-HCl (pH 7.6), 2 mM MgCl₂, 2 mM DTT, 300 μM ubiquitin (Boston Biochem), 1X ATP-Energy Regenerating Solution (ERS) (Boston Biochem), 1 μM Ube1 (E1, Boston Biochem), 10 μM UbcH5b (E2, Boston Biochem), 1 μM HDM2 (E3, Boston Biochem), and varying concentrations of peptide substrate (1 μM, 5 μM, 10 μM, 20 μM, and 30 μM) in a total reaction volume of 20 μL incubated at 30 °C for 2 hrs. The reaction was halted by the addition of 40 μL Tricine Sample Buffer (BioRad) followed by heating at 90°C for 5 min. For analysis by gel electrophoresis, samples were loaded onto SDS-PAGE gels (precast 16.5% Mini-PROTEAN Tris Tricine, Bio-Rad) using 1X tris-tricine running buffer and visualized with a Typhoon Imager (GE Healthcare Life Sciences).

Melvin A.T., Dumberger L.D., Woss G.S., Waters M.L, & Allbritton N.L. (2016). Identification of a p53-based portable degron based on the MDM2-p53 binding region. The Analyst, 141(2), 570-578.

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In vitro Ubiquitination Assay Protocol

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In vitro ubiquitination assays were performed as described previously (Saijo et al., 2003; Liu et al., 2010; Xu et al., 2014), and GST‐ABI1 and GST‐AHG3 proteins were acquired using these methods. The primers for GST‐ABI1 and GST‐AHG3 are listed in Table S1. CFP and CFP‐COP1 samples were extracted from N. benthamiana leaves in which they were transiently expressed. CFP was used as a negative control. The cell lysates were then mixed and immunoprecipitated with 15 μl anti‐GFP antibody‐conjugated agarose. The immunoprecipitated products were washed briefly with extraction buffer three times at 4°C. The ubiquitination reaction mixtures (60 μl) contained 30 ng of UBE1 (E1; Boston Biochem, Cambridge, MA, USA), 30 ng of UbcH5b (E2; Boston Biochem), 10 μg of HA‐tagged ubiquitin (HA‐Ub; Boston Biochem), 200 ng of GST‐ABI1 or GST‐AHG3, and 3 μl immunoprecipitated product in a reaction buffer containing 50 mM Tris‐HCl (pH 7.5), 10 mM MgCl₂, and 10 mM ATP. After 2 h of incubation at 30°C, the reactions were immunoblotted with anti‐GST antibody and anti‐HA antibody.

Chen Q., Bai L., Wang W., Shi H., Ramón Botella J., Zhan Q., Liu K., Yang H, & Song C. (2020). COP1 promotes ABA‐induced stomatal closure by modulating the abundance of ABI/HAB and AHG3 phosphatases. The New Phytologist, 229(4), 2035-2049.

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In vitro Ubiquitination Assay Protocol

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In vitro ubiquitination assays were performed as previously described [41 (link)], with some minor modifications. Ubiquitination reaction mixtures (60 μL) contained 30 ng of UBE1 (E1; Boston Biochem), UbcH5b (E2; Boston Biochem), and 500 ng of HA-tagged ubiquitin (HA-Ub; Boston Biochem) in a reaction buffer containing 50 mM Tris at pH 7.5, 10 mM MgCl₂, 2 mM ATP, and 0.5 mM DTT. 500 ng 6×His-TF, 500 ng 6×His-TF-COP1 (previously incubated with 20 μM zinc acetate), 500 ng 6×His-CSU2, and 500 ng 6×His-CSU2 Δcoil were applied in the reactions as indicated. After 2 h incubation at 30°C, the reactions were stopped by adding 5×sample buffer. One-half of each mixture (30 μL) was then separated onto 8% SDS-PAGE gels. Ubiquitinated TF-COP1 was detected using anti-ubiquitin (Santa Cruz), and anti-HA (Sigma-Aldrich) antibodies, respectively.

Xu D., Lin F., Jiang Y., Ling J., Hettiarachchi C., Tellgren-Roth C., Holm M., Wei N, & Deng X.W. (2015). Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association. PLoS Genetics, 11(12), e1005747.

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In Vitro Ubiquitination Assay for COP1-Mediated Protein Degradation

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In vitro ubiquitination assays were performed as described previously (19 (link), 53 (link)). Ubiquitination reaction mixtures (60 μL) contained 30 ng of UBE1 (E1; Boston Biochem), 30 ng of UbcH5b (E2; Boston Biochem), 10 μg of FLAG-tagged ubiquitin (FLAG-Ub; Boston Biochem), 200 ng of GST-WDL3, GST-WDL3 1-195, GST-HYH, or GST-WDL5, and 0.5 μg of MBP-COP1 (incubated with 20 μM zinc acetate) or bMBP-COP1 (denatured MBP-COP1 by being boiled for 10 min at 95 °C before addition to the ubiquitination reaction system) in a reaction buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl₂, and 10 mM ATP. After 2 h of incubation at 30 °C, reactions were stopped by adding 5× sample buffer. Thirty microliters of each mixture was then separated in 8% SDS/PAGE gels. Ubiquitinated GST-WDL3, GST-WDL3 1-195, and GST-HYH were detected using anti-GST and anti-FLAG antibodies (Sigma-Aldrich).

Lian N., Liu X., Wang X., Zhou Y., Li H., Li J, & Mao T. (2017). COP1 mediates dark-specific degradation of microtubule-associated protein WDL3 in regulating Arabidopsis hypocotyl elongation. Proceedings of the National Academy of Sciences of the United States of America, 114(46), 12321-12326.

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IpaH Ubiquitination Assay Protocol

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de Jong M.F., Liu Z., Chen D, & Alto N.M. (2016). Shigella flexneri suppresses NF-kB activation by inhibiting linear ubiquitin chain ligation. Nature microbiology, 1(7), 16084.

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Ubiquitination of MdCOP1 by MdBT2

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In vitro ubiquitination assay reaction mixtures (60 μL) contained 30 ng of UBE1 (E1; Boston Biochem), 30 ng of UbcH5b (E2; Boston Biochem), 10 μg of ubiquitin (Ubi; Boston Biochem), 500 ng of His-MdCOP1 (previously incubated with 20-μM zinc acetate), and 500 ng of GST-MdBT2 in reaction buffer containing 50-mM Tris-HCl (pH 7.5), 10-mM MgCl 2 , 10-mM ATP, and 2-mM DTT. The His-MdCOP1 protein that had not been incubated with zinc acetate was used as a negative control. After 2 h of incubation at 30°C, the reactions were stopped by adding loading buffer. ubiquitinated His-MdCOP1 protein was detected using anti-ubiquitin (Sigma) and anti-His (Abmart) antibodies, respectively.

Kang H., Zhang T.T., Li Y.Y., Lin-Wang K., Espley R.V., Du Y.P., Guan Q.M., Ma F.W., Hao Y.J., You C.X, & Wang X.F. (2022). The apple BTB protein MdBT2 positively regulates MdCOP1 abundance to repress anthocyanin biosynthesis. Plant physiology, 190(1).

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Analyzing Ubiquitination of MAVS by MARCH5

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Cells were treated with 10 μM of MG132 for 12 h before harvest. Whole cells were lysed with RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.1%s sodium deoxycholate, 5 mM EDTA and 5 mM EGTA) containing complete protease and phosphatase inhibitors. The same amount of protein lysates (700∼1,000 μg) was immunoprecipitated with anti-Flag or anti-MAVS antibody and further incubated with protein G agarose beads at 4 °C for 1 h 30 min. The immune complex was washed extensively four times with RIPA buffer and boiled for 5 min with 2 × sample buffer. Analysis of ubiquitylation was performed by immunoblotting using anti-Ub antibody. For in vitro ubiquitination assay, immunoprecipitated Myc-MARCH5^WT and Flag-MAVS^WT were prepared from lysates of HEK293T cells transfected with Myc-MARCH5^WT or Flag-MAVS^WT, individually. Immunoprecipitates were incubated with 100 ng of E1 (Boston Biochem), 400 ng of E2 (UbcH5b, Boston Biochem) and 2 μg of ubiquitin (Boston Biochem) in reaction buffer (50 mM Tris (pH 7.4), 2 mM MgCl₂, 4 mM ATP (Sigma), 1 mM dithiothreitol at 30 °C for 2 h, and the reaction was terminated by addition of 4 × sample buffer.

Yoo Y.S., Park Y.Y., Kim J.H., Cho H., Kim S.H., Lee H.S., Kim T.H., Sun Kim Y., Lee Y., Kim C.J., Jung J.U., Lee J.S, & Cho H. (2015). The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling. Nature Communications, 6, 7910.

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