In vitro SUMOylation assays were performed using the SUMOylation kit (Enzo Life Sciences International, Inc., Plymouth Meeting, PA, USA) according to the manufacturer’s protocol. In brief, 200 nmol/l of purified recombinant human TBL1 or TBLR1 protein was incubated with reaction mixture containing 50 mmol/ Tris-HCl (pH 7.4), 2 mmol/l DTT, 5 mmol/l ATP, 10 mmol/l MgCl2, Aos-1 (150 ng), His-Uba2 (400 ng), GST-Ubc9 (500 ng), and GST-SUMO-1 for 1 hour at 30 °C. The control reaction was carried out in the absence of ATP. After the incubation, protein SUMOylation was identified by immunoblotting using the anti-SUMO antibody provided with the kit. For in vivo assays, T24 cells were treated with WNT5a for 30 minutes, after which the nuclear extracts were prepared as described above. Extracts (200 μg of protein) were immunoprecipitated with anti-SUMO-1 antibody (Santa Cruz Biotechnology, Dallas, Texas, USA) followed by immunoblotting with anti-TBL1 or anti-TBLR1.
Sumoylation kit
Manufactured by Enzo Life Sciences
Sourced in United States
The SUMOylation kit is a laboratory tool used to study the process of SUMOylation, which is the post-translational modification of proteins by the Small Ubiquitin-like Modifier (SUMO) proteins. The kit provides the necessary components to perform in vitro SUMOylation assays, allowing researchers to investigate the effects of SUMOylation on protein function and regulation.
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Lab products found in correlation
Gradient gel, by Bio-Rad (2 mentions)
Anti sumo1 antibody, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Ubiquitination kit, by Enzo Life Sciences (1 mentions)
20s proteasome, by Enzo Life Sciences (1 mentions)
26s proteasome, by Enzo Life Sciences (1 mentions)
Sumo2, by R&D Systems (1 mentions)
Sumo1, by R&D Systems (1 mentions)
Sumo 3, by R&D Systems (1 mentions)
Senp1, by R&D Systems (1 mentions)
Ha beads, by Merck Group (1 mentions)
22 protocols using sumoylation kit
1
In vitro and in vivo TBL1/TBLR1 SUMOylation
2
HCMV UL44 SUMOylation Regulation
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293 T cells were co-transfected with pCMV-Myc-UL44 or pCMV-Myc-UL44-K410R, pcDNA-His-PIAS3 (wild-type or C334S mutant) and other SUMOylation-related plasmids (pRK-Flag-SUMO-1/-2/-3, pcDNA-HA-UBC9) as indicated using Lipofectamine 2000 (Invitrogen). After 48 h, the in vivo SUMO modifications of UL44 and its K410R mutant in transfected cells were analyzed by Western blot using anti-Myc and anti-Flag antibodies, as described in our previous work [35 (link)]. Meanwhile, to analyze the SUMOylation of UL44 under HCMV infection, U251 cells were first transfected with siControl or siPIAS3, and 24 h later infected with wt-HCMV or v-K410R at an MOI of 1. At 48 hpi (hour post infection), total cell lysates were immunoprecipitated with anti-UL44 antibody or analyzed directly by SDS-PAGE, followed by detection of UL44 and UL44-SUMO proteins using anti-UL44 or anti-SUMO-1 antibody. Additionally, in vitro SUMOylation assays were performed with the SUMOylation Kit from Enzo Life Science (Catalog #BML-UW8955) according to the manufacturer’s suggestions.
3
In vitro SUMOylation of MATα2 Protein
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Recombinant MATα2 (1 μg) was sumoylated in vitro using SUMOylation kit (Enzo Life Sciences, Farmingdale, NY) in a final volume of 10 μl containing 55 mM Tris (pH 7.5), 5.5 mM MgCl2, 2.2 mM ATP and 5.5 mM dithiothreitol. Sumoylation reactions were incubated at 37°C for 4 hours, after which proteins were analyzed by Western blotting with SUMO-1 and SUMO-2/3 antibodies.
4
In vitro SUMOylation of RAD51 by TOPORS
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In vitro SUMOylation was carried out using SUMOylation kit (BML-UW8955-0001, Enzo Life Sciences, USA) according to the manufacturer’s instructions. Briefly, 6xHis-RAD51 and GST-TOPORS were purified by a bacterial expression system. The SUMOylation reaction was performed in a 20 μl total volume with SUMO E1/E2 mix, SUMO1/2/3, 400 ng of purified GST-TOPORS and 200 nM purified 6xHis-RAD51. Reaction buffer and Mg-ATP were then added and incubated at 37°C for 1 h. The reaction was stopped by addition of 2× SDS-PAGE gel loading buffer followed by heating to 95°C for 5 min. The SUMOylation of RAD51 was showed by immunoblotting.
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In vitro SUMOylation was carried out using SUMOylation kit (BML-UW8955-0001, Enzo Life Sciences, USA) according to the manufacturer’s instructions. Briefly, 6xHis-RAD51 and GST-TOPORS were purified by a bacterial expression system. The SUMOylation reaction was performed in a 20 μl total volume with SUMO E1/E2 mix, SUMO1/2/3, 400 ng of purified GST-TOPORS and 200 nM purified 6xHis-RAD51. Reaction buffer and Mg-ATP were then added and incubated at 37°C for 1 h. The reaction was stopped by addition of 2× SDS-PAGE gel loading buffer followed by heating to 95°C for 5 min. The SUMOylation of RAD51 was showed by immunoblotting.
5
SUMOylation Assay for Viral Protein
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SUMOylation assay was carried according to a published protocol [61 (link)]. For SUMOylation of SyYVCV βC1 protein, 150 nM of substrate protein was incubated with 1 μM E1 (SAE1/SAE2), 2.5 μM E2 (ubc9) and 10 μM of 6x-HIS-tagged NbSUMO1-GG. The reaction was initiated by adding 1 mM ATP. The reaction was carried out at room temperature in a buffer containing 25 mm Tris (pH 8.0), 150 mM NaCl, 5 mM MgCl2 and 0.1% Tween 20. The reaction was terminated by adding 2X SDS lamelli buffer, followed by boiling at 95 °C for 1 min and separating on a 4–20% gradient gel. For SUMOylation assay using peptides, 5 μM of peptide substrate was used. For positive control of the reaction and reconfirmation of SUMOylation of βC1, MBP-tagged SyYVCV βC1 and SUMO domain mutants of βC1 were incubated with SUMOylation cascade enzymes as per the manufacturer’s instructions (SUMOylation kit, ENZO life sciences). HsSUMO1 was replaced with NbSUMO1. The reaction was terminated using 2x SDS non-reducing dye and the mixture was resolved in a 4–20% gradient gel (Bio-Rad), followed by detection with anti AtSUMO1 antibody.
6
SUMOylation Assay of Topoisomerase II
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SUMOylation assays were performed using a SUMOylation kit (Enzo) according to the manufacturer's instructions. Where indicated, 15 ng of human purified Topo2a (Topogen) or 100 ng of purified biotinylated peptides and 100 ng of NSE2-GST (Abnova), PIAS1-GST (Enzo) or PIAS4-GST (Abnova) were added and the reaction was incubated for 45 min at 37°C.
7
In vitro KRAS SUMOylation Assay
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In vitro SUMOylation assay was carried out at 37°C for 1 h using a SUMOylation kit (Enzo Life Science) and purified His-KRASV12 or KRASK42 proteins. For each reaction, His-KRAS or the mutant proteins (250 ng) were incubated with the following proteins: SUMO E1 (25 ng), SUMO E2 (25 ng), SUMO1 (500 ng), SUMO2 (500 ng), and/or SUMO3 (500 ng).
8
In Vitro SUMOylation Assay
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The SUMOylation kit (Enzo Life Sciences, NY, USA) was used for the in vitro SUMOylation assay. The reaction system [10 × SUMOylation buffer (2 μL), 20 × E1 activating enzyme (1 μL), 20 × E2 conjugating enzyme (1 μL), 20 × Mg-ATP (1 μL), 20 × SUMO1 (WT or mutant, 1 μL), synthesized Myc-MLK3 (390–413) (WT or K401R, 0.4 μg)] was incubated at 37 °C for 4 h [21 (link)]. Then, the reaction was boiled in Laemmli sample buffer for 5 min for analysis by immunoblotting.
9
In vitro SUMOylation Assay of β-catenin
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In vitro SUMOylation assays were conducted using the SUMOylation kit (Enzo Life Sciences, Inc.; cat. no. BML-UW8955) as per the manufacturer's protocols. In brief, the reaction was performed using β-catenin with a reaction mixture containing SUMO protein, SUMOylation enzyme, SUMOylation buffer and Mg-ATP for 1 h at 30°C as per kit protocol. After the incubation, protein SUMOylation was identified by immunoblotting using the anti-SUMO1 antibody (1:1,000 dilution) provided with the kit. The Goat anti-rabbit IgG H&L HRP conjugate secondary antibody (1:15,000 dilution; Thermo Fisher Scientific, Inc.; cat. no. 31460) was exploited in this assay at room temperature for 1 h.
10
SUMOylation Assay for SyYVCV βC1 Protein
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SUMOylation assay was carried according to a published protocol (Chatterjee et al., 2019) . For SUMOylation of SyYVCV βC1 protein, 150 nM of substrate protein was incubated with 1 uM E1 (SAE1/SAE2), 2.5 uM E2 (ubc9) and 10 uM of 6x-HIS tagged NbSUMO1-GG. The reaction was initiated by adding 1 mM ATP. The reaction was carried out at room temperature in a buffer containing 25 mm Tris (pH 8.0), 150 mM NaCl, 5 mM MgCl 2 , 0.1 % Tween 20. Reaction was terminated by adding 2X SDS lamelli buffer, followed by boiling at 95°C for 1 min and separating on a 4-20 % gradient gel. For SUMOylation assay using peptides, 5 uM of peptide substrate was used. For positive control of the reaction and reconfirmation of SUMOylation of βC1, MBP tagged SyYVCV βC1, and SUMO domain mutants of βC1 were incubated with SUMOylation cascade enzymes as per manufacturer's instructions (SUMOylation kit, ENZO life sciences). HsSUMO1 was replaced with NbSUMO1. The reaction was terminated using 2x SDS non reducing dye and the mixture was resolved in a 4-20% gradient gel (Bio Rad), followed by detection with anti AtSUMO1 antibody.
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