Standard ubiquitination reactions were performed for 60 min at 37 ° C in a total volume of 50 μl, containing 2 μg E3s (as GST-fusion proteins bound to glutathione-beads), 50 ng E1 (Enzo Life Sciences), 1 μg E2 (Enzo Life Sciences), 2 μg ubiquitin (BostonBiochem) in 50 mM Tris, pH 7.4; 150 mM NaCl; 0.5 mM dithiothreitol (DTT); 10 mM MgATP. For time-course experiments, reactions were started by the addition of ATP and stopped by the addition of 5× reducing Laemmli loading buffer (100 mM DTT final concentration) and boiling at 95 ° C for 5 min. To detect ubiquitination, reactions were separated on either a 10% acrylamide gel or a 4–12% NuPAGE (Invitrogen) and transferred to PVDF membrane for Western blotting with anti-ubiquitin antibodies.
Ubiquitin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ubiquitin is a small regulatory protein found in eukaryotic cells. It plays a central role in the post-translational modification of proteins, marking them for degradation or other cellular processes. Ubiquitin functions by covalently attaching to target proteins, thereby altering their stability, localization, or activity.
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Lab products found in correlation
Ube2r2, by R&D Systems (2 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (2 mentions)
Ube2d3, by R&D Systems (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Fluorescein isothiocyanate fitc labeled ubiquitin, by Thermo Fisher Scientific (2 mentions)
Ube2l3, by R&D Systems (2 mentions)
Ube2e1, by R&D Systems (2 mentions)
Poly ubiquitin, by Cell Signaling Technology (2 mentions)
Ube2d3, by Cell Signaling Technology (2 mentions)
Ube2r2, by Proteintech (2 mentions)
Ube2c, by Proteintech (2 mentions)
Ube2s, by R&D Systems (2 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Alexa fluor 594 donkey anti mouse, by Jackson ImmunoResearch (1 mentions)
Ubiquitin, by Merck Group (1 mentions)
Dapi nuclear stain, by Thermo Fisher Scientific (1 mentions)
400 fluorescent microscope, by Nikon (1 mentions)
Morphometric system, by Nikon (1 mentions)
14 protocols using ubiquitin
1
In Vitro Ubiquitination Assay
2
Double Immunofluorescence Staining of Tissue Slides
3
Protein Expression Analysis in Tumor Tissues
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Cells were lysed for 30 min in cold lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1% NP-40, and 0.5% sodium deoxycholate) supplemented with protease (Roche) and phosphatase (Calbiochem) inhibitors. Tumor tissues collected from mice were homogenized in 300–500 μl (five volumes) of cold lysis buffer. Equal amounts of protein (20–30 μg) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes (Whatman), and detected with antibodies against TCTP (Abcam), phospho-TCTP-Ser46, Raptor, Rictor, S6K, phospho-S6K-T389, PLK1, phospho-PLK1-Thr210, cell division cycle 25C (Cdc25C), phospho-Cdc25C-Ser198, Akt, phospho-Akt-T308, phospho-Akt-S473 (Cell Signaling Technology), phospho-S6 (eBioscience), ubiquitin, HA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Mcl-1, poly(ADP-Ribose) polymerase (PARP), p53 (Santa Cruz Biotech), and β-actin and α-tubulin (AB Frontier, Korea). The antibody information is listed in Supplementary Table S1 . To analyze the levels of multiple proteins separated on a single gel, a gel or a membrane was cut into pieces and then the membrane strips were hybridized with antibodies. Immunodetection was carried out with an enhanced chemiluminescent (ECL) kit (Perkin Elmer). Immunoreactive bands were visualized by X-ray film exposure or using Microchemi (DNR, Israel) and quantified using image analysis software (Multi-gauge v3.0, Fujifilm).
4
Antibody Detection and Visualization Protocol
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Universal type I IFN was purchased from PBL biomedical laboratories (Piscataway, NJ). Antibodies against FLAG (Sigma-aldrich), Tubulin (Sigma-Aldrich), HA (Covance, Denver, PA & Santa Cruz), β-actin (Sigma-Aldrich), UBB+1 (Millipore), GFP (Invitrogen), FK1 (Enzo life Sciences) and ubiquitin (eBioscience) were purchased from the respective manufacturers. Proteins were electroblotted onto nitrocellulose membranes (HyBond, Amersham Biosciences Inc). Rabbit anti-mouse ISG15 polyclonal antibodies have been described previously (Malakhova et al., 2003). Armenian hamster ISG15 monoclonal antibody is a kind gift from Dr. Debra Lenschow (Washington University, MA). Fluorophore conjugated secondary antibodies (Li-Cor) or horseradish peroxidase (HRP) conjugated secondary antibodies were used for detection with Odyssey system (Li-Cor) or western lighting plus-ECL system (PerkinElmer), respectively.
5
Immunocytochemical Analysis of Cellular Pathways
6
Western Blot Analysis of SIRT1 Signaling
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Cell lysates were subjected to 6–10% SDS/polyacrylamide gel followed by transfer to the polyvinylidene difluoride membranes as described previously [8 (link), 33 (link)]. The membranes were then incubated with primary antibodies against SIRT1, Snail, actin (Santa Cruz Biotechnology, Inc.; Dallas, TX, USA); ubiquitin (Life Technologies by Thermo Fisher Scientific Inc.; Waltham, MA, USA); P‐SIRT1Ser27, P‐SIRT1Ser47, P‐JNK, JNK, acetylated‐lysine, and acetylated‐p53Lys382 (Cell Signaling Technology; Danvers, MA, USA). Blots were washed with Tris‐buffered saline with 0.1% Tween‐20 and were then probed with horseradish peroxidase‐conjugated secondary antibodies (Pierce Biotechnology; Rockford, IL, USA). The transferred proteins were detected by using Western blotting detection reagents (AbClon; Seoul, South Korea).
7
Protein Extraction and Immunoblotting Protocol
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Total protein extracts were isolated with RIPA lysis buffer in the presence of a protease inhibitor mixture and phosphatase inhibitor cocktail (SIGMA). Protein extracts were quantified with the BCA Protein Assay Kit (Pierce). Equal amounts of total cellular lysates (30 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, analyzed by immunoblotting with the appropriate antibodies and then revealed by ECL (GE Healthcare). The antibodies used were as follows: ADAR1 (Santa Cruz Biotechnology), ADAR1 (Bethyl), CDK2 (Santa Cruz Biotechnology), YTHDF1 (Abcam), METTL3 (Abcam), METTL14 (Bethyl), cyclinE (Santa Cruz Biotechnology), p57 (Santa Cruz Biotechnology), Skp2 (Santa Cruz Biotechnology), CDC14B (LifeSpan), ADAR2 (Santa Cruz Biotechnology), Ubiquitin (Thermo Fisher), β-actin (Santa Cruz Biotechnology), GAPDH (Cell Signaling), and the anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology).
8
Ubiquitin Buffer Exchange Protocol
9
Recombinant Protein Expression in E. coli
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For recombinant protein expression using Escherichia coli, and for cell-free synthesis-coupled transcription–translation, the target cDNA fragments were sub-cloned into the pCR2.1-TOPO vector (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Proteins, subjected to affinity purification, were N-terminally fused with a modified natural poly-histidine N11-tag (amino acid sequence: MKDHLIHNHHKHEHAHAEH) or a maltose-binding protein (MBP) tag, as well as with a TEV (tobacco etch virus) protease recognition site and a GSSGSSG linker sequence [21 (link)]. These tags were introduced using TOPO cloning. In the cell-free synthesis system, proteins, tagged with either the N11-tag or the MBP tag, were designed to have the same protein sequence if the tag was to be removed using TEV. For baculovirus-insect cell expression, we used the Bac-to-Bac Baculovirus Expression System (Thermo Fisher Scientific).
cDNAs were derived from the original clone sets as follows: ubiquitin from a clone set obtained from the Institute of Medical Science, the University of Tokyo; UBE2D1 from a clone set purchased from Thermo Fisher Scientific; UBA1 from a clone set purchased from OriGene Technologies (Rockville, MD, U.S.A.); and BRAP from a clone set obtained from the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan).
cDNAs were derived from the original clone sets as follows: ubiquitin from a clone set obtained from the Institute of Medical Science, the University of Tokyo; UBE2D1 from a clone set purchased from Thermo Fisher Scientific; UBA1 from a clone set purchased from OriGene Technologies (Rockville, MD, U.S.A.); and BRAP from a clone set obtained from the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan).
10
Ubiquitin Conjugation Pathway Characterization
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Ubiquitin, Fluorescein isothiocyanate (FITC)-labeled Ubiquitin, and ATP were purchased from ThermoFisher. UBE2D3, UBE2R2, UBE2S, UBE2L3, and UBE2E1 were purchased from R&D Systems. Primary antibodies for UBA1a/b (Cell Signaling, 4891S), Poly-Ubiquitin (Cell Signaling, 3936S), UBE2C (Proteintech, 66087-1-Ig), UBE2D3 (Cell Signaling, 4330S), and UBE2R2 (Proteintech,14077-1-AP), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:1000. HRP-conjugated antibodies were visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500).
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