DNA fragment coding full-length of Oct-1 was subcloned to the pET28a vector (pET28a-Oct-1). The plasmid was confirmed by DNA sequencing. TNT T7 Quick System Kit (Promega) was used to perform in vitro transcription and translation (IVTT) with linearized pET28a-Oct-1. The reaction was incubated at 30 °C for 16 h. Western blot was conducted to determine IVTT efficiency. Reaction mixture without linearized pET28a-Oct-1 was used as control. The in vitro transcription and translation product was used as substrate to perform in vitro ubiquitination assay. The IVTT product was incubated with 0.05 μM recombinant E1 enzyme UBE1(E-304, Boston Biochem), 0.5 μM recombinant E2 enzyme UBCH5a and UBCH5b (E2-616 and E2-622, Boston Biochem) and 5 μM ubiquitin protein (U-100H, Boston Biochem) with or without addition of 0.5 or 1.0 μg TRIM21 protein (Creatvie-Biomart) and 1 mM ATP in a total volume of 20 μl (30 °C, 16 h). Western blot using anti-Oct-1 and anti-ubiquitin antibodies was performed to detect protein ubiquitination.
Ubch5b
Manufactured by R&D Systems
Sourced in United States
UbcH5b is an E2 ubiquitin-conjugating enzyme that functions in the ubiquitin-proteasome pathway. It catalyzes the covalent attachment of ubiquitin to target proteins, marking them for degradation by the 26S proteasome.
Automatically generated - may contain errors
Lab products found in correlation
Ubiquitin, by R&D Systems (5 mentions)
Mg atp, by R&D Systems (4 mentions)
Ubch5c, by R&D Systems (2 mentions)
Flag ubiquitin, by R&D Systems (2 mentions)
E1 ube1, by R&D Systems (2 mentions)
Recombinant ubiquitin, by R&D Systems (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Fluorescent ub, by R&D Systems (2 mentions)
Typhoon fla 9500, by GE Healthcare (2 mentions)
E3 ligase reaction buffer, by R&D Systems (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Flag antibody, by Merck Group (1 mentions)
Glutathione sepharose beads, by Cytiva (1 mentions)
His6 ubiquitin, by Merck Group (1 mentions)
Energy regeneration solution, by R&D Systems (1 mentions)
Ha ubiquitin, by R&D Systems (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Ubiquitin, by Merck Group (1 mentions)
Anti ubiquitin, by Merck Group (1 mentions)
22 protocols using ubch5b
1
In Vitro Ubiquitination of Oct-1 Transcription Factor
2
DEK Ubiquitylation by SPOP-Cul3 Complex
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Each reaction was performed with 100 nM DEK (OriGene) in 20 μL of ubiquitylation buffer (50 mM Tris-HCl, 200 mM NaCl, 1 mM DTT, 10 mM MgCl2) containing 4 mM ATP, 5 nM SPOP (Novus Biologicals), 10 nM Cul3/Rbx1 (Ubiquigent), 500 nM UbcH5b, 100 nM UbE1 (Boston Biochem, Inc.), and 25 μM ubiquitin (Boston Biochem, Inc.), without or with various concentration (0.25, 0.5, and 1 nM) of G3BP1 (Prospec). The reactions were incubated at 37 °C for 1 h and stopped by the addition of SDS sample buffer. Samples were separated by 10% SDS–PAGE and analyzed for the respective antibodies mentioned in the figure.
3
In Vitro Ubiquitination Assay
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Ubiquitination reactions were carried out in a 20-μl mixture containing buffer A (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM DTT), 2 μg of HA-tagged ubiquitin, 0.25 μg of E1 (Boston Biochem, Cambridge, MA, USA) and 1 μg of E2 (human recombinant UbcH5b from Boston Biochem) in the presence or absence of ubiquitin ligases and substrates. Reactions were incubated at 37°C for 1 h with shaking and stopped by adding an equal volume of Laemmli sample buffer 4X containing 100 mM DTT and boiling. Some reactions were carried out with GST fusion proteins bound to glutathione-agarose beads, and the beads were washed five times with NP40 buffer before boiling in Laemmli sample buffer with 100 mM DTT.
4
In Vitro Ubiquitination Assay of GID Complex
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500 nM Hbp1-FLAG, 100 nM UbE1 (Boston Biochem), 1 μM E2 enzyme (Cdc34b, UbeH2, UbcH5c (Boston Biochem), UbcH5b, Ube2G2, Ube2E2), 50 μM Ubiquitin and 500 nM FLAG-GID complex were incubated at 37°C in a total volume of 50 μl reaction buffer (50 mM Tris pH 7.6, 3 mM ATP, 0.5 mM DTT, 10 mM MgCl2). At the indicated time points, 10 μl of the reaction mixture was analyzed by SDS-PAGE and immunoblotting. For native HSS-GID particles we performed immunoprecipitation experiments and ubiquitination assays as described above with the following modifications; 25 μl of HSS-GID eluate (GID2 or GID5) was incubated with 100 nM UbE1, 1 μM E2 enzyme, 15 μM Ubiquitin and in a total of 50 μl E3 reaction Buffer. 10 μl samples were taken at variable time points and autocatalytic activity of the GID complex analyzed by Western Blotting.
5
Ubiquitin Modification and CIITA Regulation
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Flag-K141R, K144R, K141/144R, and Myc-CIITA were kindly provided by Dr. Jenny Ting. Flag-pCAF was a generous gift from Drs. Linares et al. [23 (link)]. Myc-pCAF was subcloned into Myc tagged pCMV-3 using the EcoR1 restriction site. HA-K48 Ub and K63 Ub were a gift from Dr. Ted Dawson. Both, HA-K48 and K63 ubiquitin have all internal lysine residues of ubiquitin mutated to arginine except K48 or K63, allowing polyubiquitination to only occur on those lysine residues. The HLA-DRA luciferase reporter construct was described previously [27 (link)]. The E1 activating enzyme UBE1 (Boston Biochem, Boston, MA), E2 conjugating enzyme UbcH5b (Boston Biochem), Flag ubiquitin (Boston Biochem), Hdm2 (Boston Biochem), and His-pCAF (Proteinone, Rockville, MD) were all obtained commercially.
6
In Vitro CIITA Ubiquitination Assay
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The CIITA in vitro ubiquitination assay was carried out in 150 μL of reaction mixture containing 40 mM Tris-HCL (pH 7.5), 5 mM MgCl2, 2 mM dithiothreitol, 1 mM Creatine Phosphate, 2 mM ATP, 400 ng of Recombinant GST-CIITA (substrate), 400 ng of GST-Hdm2 (Boston Biochem), 400 ng Recombinant His-pCAF (E3 ligase candidate) (Proteinone Rockville, MD), 500 ng Flag ubiquitin (Boston Biochem, Boston, MA), 200 ng E1 activating enzyme, UBE1 (Boston Biochem), 200 ng E2 conjugating enzyme, and UbcH5b (Boston Biochem). All components were added and incubated at 37°C for 60 minutes and were analyzed via SDS-PAGE. Ubiquitination was detected by immunoblot using Flag antibody (Sigma), and CIITA ubiquitination was verified with GST (Abcam), and pCAF was verified with monoclonal α pCAF antibody (Santa Cruz). Verification of Hdm2 ubiquitination was detected using GST (Abcam).
7
Ubiquitination assay of p73 and ΔNp73
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The reactions were carried out at 30°C for 15 min in 25 μl of ubiquitylation reaction buffer (40 mM Tris-HCl at pH 7.6, 2 mM dithiothreitol [DTT], 5 mM MgCl2, 0.1 M NaCl, 2 mM ATP) containing the following components: 100 μM ubiquitin, 20 nM E1 (UBE1), and 100 nM UbcH5b (all from Boston Biochem); bacterially purified MBP-WWP2 and MBP-WWP1 E3 ligases were added to the reaction mixture. Bacterially purified GST, GST-p73, and GST-ΔNp73 bound to glutathione-Sepharose beads (Amersham) were used as substrates in the reaction mixture. After the reaction, beads were washed three times with NETN buffer and boiled with SDS-PAGE loading buffer; ubiquitination of substrates was detected by Western blotting with anti-GST antibody.
8
In Vitro Ubiquitination Assay for GST-hMEX-3C
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For this assay, 1 μg of bacterially produced GST‐hMEX‐3C Ring finger domain WT and mutants were, respectively, incubated at 37°C for 90 min in a reaction mixture with 100 nM E1 (Sigma‐Aldrich), 1 μM each of the different E2s (UbcH5b, UbcH5c, and UBE2H, Boston Biochem), 5 μM His6 ubiquitin (Sigma‐Aldrich), 5 mM ATP, 5 mM MgCl2, 5 mM DTT, and 50 mM Tris–HCl (pH 7.5) and ddH2O to a final volume of 10 μL. After dissolved the reactions with 10 μL of 2 × SDS sample loading buffer, products were analyzed by SDS‐PAGE. The reactions were analyzed by western blotting using anti‐Ub and anti‐GST antibodies (Sigma‐Aldrich).
9
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).
10
In vitro Ubiquitylation of TRIAD3 Proteins
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In vitro-ubiquitylation samples contained 10 mM ATP, 15 mM ubiquitin (Sigma-Aldrich), 0.6 µM UbcH5b (BostonBiochem®), 0.1 µM E1 (BostonBiochem®), and 1 µM of recombinant TRIAD3 proteins or wild type and mutant TRIAD3B immobilised to anti-V5 agarose beads in a buffer of 20 mM Hepes (pH 8.0), 150 mM NaCl, 10 mM MgCl, and 0.5 mM DTT. Ubiquitylation reactions were carried out for 90 min at 37 °C and stopped for western blot analysis by the addition of Laemmli sample buffer.
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