For in vitro ubiquitination, 400 ng of purified Flag-PTEN from 293 cells was incubated with 40 ng E1 (UBE1) (E-305 Boston Biochem), 500 ng E2 (UbcH5a) (E2–616 Boston Biochem), 10 μg His-Ub variants (Boston Biochem), 8 mM ATP, 1× ligase reaction buffer [Fisher Scientific (Thermo Fisher Scientific) # B71], and 1× Energy regeneration system (Boston Biochem #B-10), 400 ng NEDD4 (Sigma Aldrich # SRP0226), and 400 ng WWP1 (Sigma Aldrich # SRP0229) at 37°C for 1 hour in 25 μl of reaction mixture.
E2 ubch5a
Manufactured by R&D Systems
Sourced in United States
E2 (UbcH5a) is a recombinant human ubiquitin-conjugating enzyme that catalyzes the conjugation of ubiquitin to target proteins. It functions as part of the ubiquitin-proteasome system, which is involved in the regulation of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Ubiquitin aldehyde, by R&D Systems (4 mentions)
Ubiquitin, by Merck Group (4 mentions)
E1 uba1, by R&D Systems (3 mentions)
Ubiquitin, by R&D Systems (3 mentions)
His6 ubch3 e2, by R&D Systems (3 mentions)
E1 ube1, by R&D Systems (2 mentions)
Amersham 600 ccd based imaging system, by GE Healthcare (2 mentions)
His ub variants, by R&D Systems (1 mentions)
Nedd4, by Merck Group (1 mentions)
L1170, by Promega (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Atp bioluminescence assay kit hs 2, by Roche (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti ha, by Merck Group (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Gst agarose beads, by Merck Group (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
14 protocols using e2 ubch5a
1
In Vitro Ubiquitination of PTEN
2
Assaying C/EBPβ Ubiquitination In Vivo and In Vitro
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In vivo and in vitro ubiquitination assay were performed as previously described [60 (link)]. For C/EBPβ in vivo ubiquitination assay, the BV2 cells or 293T cells that transfected with the indicated expression vectors were treated with MG132 for 4 hours and then were lysed with lysis buffer containing protease inhibitors and N-ethylmaleimide (Sigma). The cell extracts were boiled for 5 minutes in the presence of 1% SDS to dissociate the C/EBPβ-interacting proteins and then were diluted with lysis buffer until the concentration of SDS was 0.1% before immunoprecipitation. C/EBPβ was then immunoprecipitated from the cell extracts and was assessed the status of ubiquitination with anti-ubiquitin antibody. For C/EBPβ in vitro ubiquitination assay, the HA-Peli1, HA-Peli1ΔC, and C/EBPβ were translated in vitro by using cell-free quick coupled transcription/translation system (L1170, Promega). The ubiquitination reaction was processed by mixing the translated proteins with precharged ubiquitin-conjugating enzyme E2 UbcH5a (E2-800, Boston Biochem) and incubating at 37°C for 4 hours according to the manufacturer’s protocol. The reaction was then terminated by boiling for 5 minutes in SDS loading buffer, and the samples were subjected to SDS-PAGE and immunoblotting to detect the ubiquitination of C/EBPβ mediated by Peli1.
3
In vitro ubiquitination of CDK12-cycK
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In vitro ubiquitination was performed by mixing N8DDB1-CUL4-RBX1 at 70 nM with a reaction mixture containing kinase inhibitors at 2 μM, CDK12-cycK at 500 nM, E1 (UBA1, BostonBiochem) at 50 nM, E2 (UBCH5a, BostonBiochem) at 1 μM, and ubiquitin at 20 μM. Reactions were carried out in 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 0.2 mM CaCl2, 1 mM ATP, 0.1% Triton X-100 and 0.1 mg/mL BSA, incubated for 0–30 minutes at 30°C and analysed by western blot using anti-cycK and anti-rabbit IgG antibodies. Blots were scanned on an Amersham 600 CCD-based imaging system (GE Life Sciences).
4
Assay for CBP E3 Ligase Activity
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To determine CBP E3 ligase activity, CBP was immunoprecipitated from cytoplasm or nuclear fractions diluted with high salt buffer [10mMHEPES(pH7.5), 150mM NaCl, 150mMKCl, 1mMMgCl2, 0.5%Triton X-100, supplemented with protease inhibitors), using A-22 antibody and protein A Sepharose. The IPs were washed in high salt buffer three times followed by two washes in Ub buffer (25mMHEPES, pH7.4,10mMNaCl, 3 mM MgCl2, 0.05% Triton X-100, 0.5 mM DTT, 3 mM Mg-ATP). The washed and equilibrated IPs were then incubated with 100 ng E1 (rabbit; Boston Biochem), 25ng E2 (UbcH5a, human recombinant; Boston Biochem), and 5μg Ub (human recombinant; Boston Biochem) for 60 min at 37°C. Reactions were then stopped by the addition of LDS sample buffer, followed by SDS-PAGE and immunoblotting.
5
In vitro Ubiquitination of ZFP91
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In vitro ubiquitination was performed by mixing ZFP91 at 0.6 μM, and N8CRL4CRBN at 80 nM with a reaction mixture containing IMiDs at indicated concentrations or a DMSO control, E1 (UBA1, Boston Biochem) at 40 nM, E2 (UBCH5a, Boston Biochem) at 1.2 μM, ubiquitin at 23 μM. Reactions were carried out in 50 mM Tris pH 7.5, 30 mM NaCl, 5 mM MgCl2, 0.2 mM CaCl2, 2.5 mM ATP, 0.1% Triton X-100 and 0.1 mg ml−1 BSA, incubated for 15–30 min at 30 °C and analysed by western blot using anti-ZFP91 and anti-rabbit IRDye 800CW antibodies. Blots were scanned on a LICOR Odyssey infrared imaging system (uncropped immunoblots are provided in Supplementary Fig. 4 ).
6
Assay for CBP E3 Ligase Activity
7
In Vitro ABCE1 Ubiquitination Assay
8
In vitro ubiquitination of CDK12-cycK
9
Lysine to Arginine Mutagenesis and Ubiquitylation Assay
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Plasmids containing the DAX1 or DPPA5 gene were isolated and subjected to site-directed mutagenesis using the New England Biolabs Q5 Site-Directed Mutagenesis Kit (E0554S) to introduce lysine to arginine mutations. Reactions were performed per manufacturer’s protocol. HEK293T cells were transfected with plasmids encoding Flag-FBXO9; Flag-ΔFBXO9; Flag-FBXO9 ΔTPR; HA-DPPA5; HA-DPPA5 K9,16R; HA-DPPA5 K35R; and HA-DPPA5 K103,109R. Forty-eight hours post transfection, cells were immunopurified from the whole cell extracts using Anti-HA (Sigma) or Anti-Flag (Sigma) beads overnight at 4 °C. The immunopurified E3 ligase (0.5 µg) proteins were incubated with immunopurified 0.5 µg substrate, E1 (Boston Biochem), E2-UbcH5a (500 ng, Boston Biochem), ubiquitin (0.5 µg, Boston Biochem), and in the presence or absence of activated ATP (10 mM). Ubiquitylation reactions were performed in assay buffer (100 mM NaCl, 1 mM DTT, 5 mM MgCl2, 25 mM Tris-Cl (pH 7.5)) and incubated at 30 °C for 2 hours. The reactions were stopped with 2× laemmli buffer (10 minutes at 95 °C), resolved on SDS-PAGE gels, and analyzed by Western blot.
10
In Vitro Ubiquitination Assay
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The GST-CRY1-WT and GST-CDT1 substrates were captured and eluted off of GST-agarose beads (Sigma) after IPTG induction in transformed BL21 cells for 3 hr at 37°C. The FLAG-tagged DDB1 and CUL4A E3 complexes were immunoprecipitated from U2OS cells by anti-FLAG M2 antibody (Sigma) after Ad-Ddb1/Cul4A transduction. Ad-GFP transduced cells were used as IP control. Protein-A sepharose beads were equilibrated in ligase assay buffer (25 mM Tris-HCl pH = 7.5, 50 mM NaCl, 1 mM EDTA, 0.01% NP-40, 10% glycerol) twice and mixed with the GST substrate in 30 μL of the ubiquitin mix containing: 25 mM Tris-HCl, pH = 7.4, 5 mM MgCl2, 2 mM ATP, 2 mM sodium fluoride, 1mM DTT, 10 nM okadaic acid, 250 μM ubiquitin aldehyde (Boston Biochem), 120 ng E1 (His6-UBE1, Boston Biochem), 300 ng E2 (UbcH5a, Boston Biochem), and 10 μg ubiquitin (Boston Biochem) [22 (link)]. The reactions were kept in a 37°C shaker for 2 hr and then denatured by adding 5X SDS loading buffer and boiling at 95°C for 5 mins. The final reactions were run in an 8% SDS-PAGE gel and subjected to immunoblotting with anti-ubiquitin antibody.
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