Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were mixing with 20X E1 Enzyme, 10X Mg2+-ATP Solution, 10X Ubiquitin Solution, 1ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer. The reaction was carried out at 37 °C for 1 h and products were analyzed by western-blot assays with anti-YAP antibody (#14074, Cell signaling, 1:1000).
Ubiquitination kit
Manufactured by R&D Systems
Sourced in United States
The Ubiquitination Kit is a laboratory tool designed to facilitate the study of ubiquitination, a process where ubiquitin molecules are attached to proteins, marking them for degradation or other cellular functions. The kit provides the necessary reagents and components to perform ubiquitination assays in a controlled experimental setting.
Automatically generated - may contain errors
Lab products found in correlation
Tnt quick coupled transcription translation system, by Promega (5 mentions)
E1 enzyme, by R&D Systems (3 mentions)
Ube2d1, by Sino Biological (3 mentions)
E2 enzyme, by R&D Systems (3 mentions)
Anti yap antibody, by Cell Signaling Technology (1 mentions)
Tnt quick coupled transcription and translation system, by Promega (1 mentions)
Anti p53 antibody, by Santa Cruz Biotechnology (1 mentions)
Ubch7, by R&D Systems (1 mentions)
12 protocols using ubiquitination kit
1
Ubiquitination Assay Protocol
2
In Vitro and In Vivo p53 Ubiquitination Assays
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In vitro ubiquitination assays using a ubiquitination kit (Boston Biochem, MA, USA) following the manufacturer’s protocol. Briefly, preparing a mix in 1.5 ml tubes and following the volumes: 7 μl dH2O, 2 µl 10× reaction buffer, 2 µl 10× ubiquitin, 1 µl 20× E1 Enzyme, 2 μl 10× E2 conjugating enzyme, 0.5 μg His-TRIM31, 1 μg GST-p53. Adding 2 µl of 10× Mg2+-ATP solution in a final volume of 50 μL to initiate a conjugation reaction. Mixed by pipetting or gently flicking tube, and incubate for 3 h in 37 °C water bath. The reaction was terminated by 1× SDS-PAGE loading buffer. The ubiquitination of GST-p53 was detected by western blot with an anti-p53 antibody.
In vivo ubiquitination assays the ubiquitination of endogenous p53, after transfection with various plasmids for 48 h, the MCF7 cells were treated with MG132 (20 μM) for 4 h before harvest. The cell lysates were prepared and incubated with 1 μg of anti-p53 antibody overnight at 4 °C. The next day, added in 20 μl proteinA/G beads (MCE, New Jersey, USA) at 4 °C for 4 h. Washing the beads with TBS buffer 3 times. The immunoprecipitates were added in 1× SDS Loading Buffer and further assay by western blot.
In vivo ubiquitination assays the ubiquitination of endogenous p53, after transfection with various plasmids for 48 h, the MCF7 cells were treated with MG132 (20 μM) for 4 h before harvest. The cell lysates were prepared and incubated with 1 μg of anti-p53 antibody overnight at 4 °C. The next day, added in 20 μl proteinA/G beads (MCE, New Jersey, USA) at 4 °C for 4 h. Washing the beads with TBS buffer 3 times. The immunoprecipitates were added in 1× SDS Loading Buffer and further assay by western blot.
3
TRIM26 Regulation of IRF3 Binding and Ubiquitination
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IRF3, TRIM26 WT and C16/36A mutant proteins were expressed with a TNT Quick Coupled Transcription/Translation System (Promega) according to the instructions of the manufacturer. Binding assays were performed by mixing TRIM26 and IRF3 together, followed by IP with TRIM26 antibody and WB with IRF3 antibody. Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer.
4
Analyzing Protein Ubiquitination in HEK293 Cells
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The proteins were over-expressed in HEK293 cells and immunoprecipitated with antibodies. Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were mixing with 20X E1 Enzyme, 10X Mg2 + -ATP Solution, 10X Ubiquitin Solution, 1ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 ul reaction buffer. The reaction was carried out at 37 °C for 1 h and products were analyzed by western-blot assays with anti-TAZ antibody.
5
In vitro MG53 and RAC1 Ubiquitination
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MG53 and RAC1 proteins were expressed in vitro by a TNT Quick Coupled Transcription and Translation System (Promega, Madison, WI, USA) according to the manufacturer’s protocol. In vitro ubiquitination assay was performed with a ubiquitination kit (Boston Biochem, MA, USA) following the manufacturer’s recommended protocol. The MG53 and RAC1 proteins were expressed in vitro separately and these two reaction mixtures were incubated in the Ubiquitin Conjugation Reaction Buffer (# B-70) containing ubiquitin (# Ue100H), UBE1 (# E1-305), UbcH5a (# E2-616), ATP and MgCl2 at 30 °C for 60 min.
6
In Vitro TRAF3-Mediated Ubiquitination
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Recombinant TRAF3, Triad3A, iOPN-WT, iOPN-N, iOPN-C proteins were made with a TNT Quick Coupled Transcription/Translation System (Promega) according to the instructions of the manufacturer. Briefly, reaction mixture contained 1ug indicated plasmid DNA and 1mM methionine was incubated at 30 °C for 60–90 minutes, and WB was performed for analyzing the results of translation. Binding assays were performed by mixing TRAF3, Triad3A and iOPN-WT protein together, followed by IP with TRAF3 antibody and WB with OPN antibody. Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were mixing with 100 nM E1, 2 mM E2 and 200 mM Ub-K48 in a final volume of 20 ml reaction buffer (50 mM Hepes pH 8.0, 100 mM NaCl, 10 mM Mg2+ −ATP, 0.5 mM DTT). The reaction was carried out at 37 °C for 1 h and products were analyzed by immunoblotting with anti-TRAF3 antibody.
7
TRIM31 and NLRP3 Protein Binding Assay
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NLRP3, TRIM31 WT and ΔRING mutant proteins were expressed with a TNT Quick Coupled Transcription/Translation System (Promega) according to the instructions of the manufacturer22 (link). Binding assays were performed by mixing Flag-tagged TRIM31 and NLRP3 together, followed by IP with Flag antibody and WB with NLRP3 antibody. Ubiquitination was analysed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer.
8
Ubiquitination Assay for YAP, FBXW7, and OTUB1
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YAP and FBXW7 were cloned into the pET26b vector. OTUB1 were cloned into the pGEX-4T1 vector. Protein purification followed the general procedure in the pET System Manual. Ubiquitination was performed with the ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were incubated with 20X E1 Enzyme, 10X Mg2+-ATP Solution, 10X Ubiquitin Solution, 1 ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer at 37 °C for 1 h. Finally, products were analyzed by western-blot assays.
9
Protein Interactions and Ubiquitination
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Trim31, Rhbdf2, and Trim31 with RING domain deletion proteins were expressed with a TNT® Quick Coupled Transcription/Translation System (Promega) in accordance with the previous reports and manufacturer’s instructions15 (link),22 ,25 (link). Protein interaction binding assays were performed by mixing corresponding Flag-tagged Rhbdf2 and Trim31 together, followed by immunoprecipitation with Flag antibody and immunoblotting with Trim31 antibody. Ubiquitination levels were analyzed with a ubiquitination kit (Boston Biochem) following protocols of the manufacturer’s instructions.
10
Ubiquitination of P53 by MDM2
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Ubiquitination was analyzed with a ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. The recombinant proteins MDM2 and P53 were acquired from Sangon Biotech (China). The RNF187 protein was expressed in and purified from HEK293 cells with anti-Myc beads. Recombinant proteins were mixed with 20X E1 enzyme, 10X Mg2+-ATP solution, 10X ubiquitin solution, 1 μg E2 enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer. The reaction was carried out at 37 °C for 1 h, and the products were analyzed by western blotting with an anti-P53 antibody (Santa Cruz, SC126).
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