The chymotrypsin-like peptidase activity of purified proteasomes was measured with 100 μM Suc-LLVY-AMC (Enzo) in a buffer containing 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM ATP, 1 mM DTT, and 0.05 mg/mL BSA. When indicated, 0.1 mM ATPᵧS (Sigma) or 1 μM ubiquitin aldehyde (Boston Biochem) was added to the reaction buffer. Fluorogenic hydrolysis was measured at excitation=380 nm and emission=460 nm at 37°C for 60 minutes in a BioTek Synergy plate reader. Rate of hydrolysis was calculated based on the slope of fluorescence increase over time during the linear phase of the reaction.
Ubiquitin aldehyde
Manufactured by R&D Systems
Sourced in United States
Ubiquitin aldehyde is a laboratory reagent used in research. It is a synthetic derivative of the ubiquitin protein, which plays a central role in the ubiquitin-proteasome system. Ubiquitin aldehyde functions as an inhibitor of deubiquitinating enzymes, which are responsible for removing ubiquitin tags from proteins.
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Lab products found in correlation
Ubiquitin, by Merck Group (4 mentions)
E2 ubch5a, by R&D Systems (4 mentions)
Ubiquitin, by R&D Systems (3 mentions)
His6 ubch3 e2, by R&D Systems (3 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (2 mentions)
Creatine phosphate, by Merck Group (2 mentions)
Myc ubiquitin, by R&D Systems (2 mentions)
Ubch5c, by R&D Systems (2 mentions)
Synergy plate reader, by Agilent Technologies (1 mentions)
Suc llvy amc, by Enzo Life Sciences (1 mentions)
E1 enzyme, by R&D Systems (1 mentions)
Creatine phosphokinase, by Merck Group (1 mentions)
E2 enzyme, by R&D Systems (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Glutathione agarose beads, by Thermo Fisher Scientific (1 mentions)
Ubch5b e2, by R&D Systems (1 mentions)
Mg132, by R&D Systems (1 mentions)
Ha ubiquitin, by R&D Systems (1 mentions)
Ubiquitin antibody, by Santa Cruz Biotechnology (1 mentions)
Ucp2 antibody, by Santa Cruz Biotechnology (1 mentions)
20 protocols using ubiquitin aldehyde
1
Proteasomal Chymotrypsin-like Activity Assay
2
In Vitro Ubiquitination Assay of DCX
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In vitro ubiquitination assays were done as previously described (68 (link)), but modified for 48-well plates. Briefly, the ubiquitination reaction mixture contained 125 ng of E1 enzyme (Boston Biochem), 250 ng of E2 enzyme (Boston Biochem), immunopurified Cul3–KLHL15 E3 complex, 10 μg of Myc-ubiquitin (Boston Biochem), 1 μM ubiquitin aldehyde (Boston Biochem), 10 mM MgCl2, 2 mM DTT, 10 mM creatine phosphate (Sigma), 0.5 mg/ml creatine phosphokinase (Sigma), and 20 μg of purified proteins including DCX-HaloTag-His6 (WT or Y259L) or Halo-His6 as control protein. The ubiquitination reactions were diluted to a final volume of 0.25 ml in 50 mM HEPES, pH 7.5, initiated upon addition of 5 mM ATP (Thermo Scientific), and incubated for up to 2 h at 37 °C on a titer plate shaker (constant speed 3, Lab-Line Instruments, Inc). Thirty-microliter reaction mixtures were sampled at the indicated times and immediately terminated in 4× Laemmli buffer. Proteins were then resolved on 8% gels by SDS-PAGE, and ubiquitinated proteins were detected by immunoblotting with Myc-Tag antibody.
Relative DCX ubiquitination in vitro was quantified by dividing Myc-Tag signals by DCX signals and GFP (KLHL15) signals in the same lane and normalizing to the zero time point (set to 1). The enzymatic kinetics was plotted in Michaelis–Menten model using GraphPad Prism.
Relative DCX ubiquitination in vitro was quantified by dividing Myc-Tag signals by DCX signals and GFP (KLHL15) signals in the same lane and normalizing to the zero time point (set to 1). The enzymatic kinetics was plotted in Michaelis–Menten model using GraphPad Prism.
3
Purification and Ubiquitination of APC
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APC was purified by immunoprecipitation of Cdc27 from Xenopus interphase egg extract using Protein G Sepharose beads (GE Healthcare Life Sciences, Pittsburgh, PA) and anti-Cdc27 antibodies (AF3.1; Santa Cruz Biotechnology, Dallas, TX) as previously described (Wei et al., 2004 (link)). For each ubiquitination reaction, 5 µl of APC-bound beads was incubated with 0.75 µM purified E1 (Boston Biochem, Cambridge, MA), 2 µM His-UbcH10 (Boston Biochem), 7.5 mg/ml ubiquitin (Boston Biochem), 0.5 µl 20× Energy Regeneration Mix (2 mg/ml creatine phosphokinase, 20 mM ATP, 200 mM Creatine Phosphate, 20 mM HEPES, 20 mM MgCl2, 0.1% BSA), 5 µM ubiquitin aldehyde (Boston Biochem), and 10 mM DTT. 1 µl of in vitro transcription/translation reaction product and 0.4 nM His-Cdh1 or equal volume of Cdh1 dialysis buffer was incubated in each reaction for 90 minutes. Reaction products were separated by SDS-PAGE and visualized by autoradiography.
4
Smad2 Ubiquitination Profiling Protocol
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Cells were harvested in non-denaturing lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8.0, 1 mM EGTA, pH 8.0 and 0.5% Nonidet-P40) supplemented with phosphatase and protease inhibitors as per immunoblotting. Lysates were additionally supplemented with 2 μM ubiquitin aldehyde (Boston Biochem) to prevent the action of deubiquitinating enzymes. Protein within lysates were purified and quantified, with 1 mg used in the subsequent immunoprecipitation with Smad2 antibody for 90 min at 4 °C with rotation. Immunocomplexes were isolated from the lysates using Protein-G conjugated Dynabeads through a further 1 h incubation under the same conditions. Beads were then washed 3 times with cold non-denaturing lysis buffer to remove nonspecific binding prior to protein elution with 2 × Laemmli buffer. Elutes were subjected to immunoblotting and the degree of ubiquitination was assessed using an anti-ubiquitin antibody.
5
In vitro Nedd4-Pax7 Ubiquitination Assay
6
UCP2 Ubiquitination Assessment Protocol
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To examine the ubiquitination status of UPC2, the immunoprecipitation (IP) for UCP2 was conducted first as previously described with modifications (20 (link)). Briefly, the HUVECs subjected to the indicated treatment were lysed with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) followed by the addition of ubiquitin aldehyde (Boston Biochem, Inc., Cambridge, MA, USA), a specific inhibitor of ubiquitin C-terminal hydrolases, at a final concentration of 2.53 µM. The lysate was clarified by centrifugation at 14,000 × g for 5 min at 4°C. Following centrifugation, the cell lysate was incubated with UCP2 antibody (Santa Cruz Biotechnology, Inc.) followed by incubation with protein G-agarose (KPL, Inc., Gaithersburg, MD, USA). To detect the ubiquitination UCP2, The IP samples containing UCP2 were subjected to western blot analysis with ubiquitin antibody (Santa Cruz Biotechnology, Inc.).
7
In vitro Ubiquitylation of SuFu Protein
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In vitro ubiquitylation was performed as previously described64 (link),65 (link). In vitro translated protein SuFu, produced using TnT® Coupled Wheat Germ Extract System (Promega, Madison, WI, USA), was incubated at 30 °C with GST or Itch-GST or Itch-GST and βarr2-GST 400ng (Abnova, Heidelberg, Germany), 50 mM Tris-HCl at pH 7.5, 5 mM MgCl2, 200 µM okadaic acid, 2 mM ATP, 0.6 mM DTT, 1 mM ubiquitin aldehyde, E1, UbcH7, and either wild-type or mutant ubiquitin (Boston Biochem). Polyubiquitylated products were subjected to SDS-PAGE and detected by fluorography.
8
FBXO38 Ubiquitination Assay
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HEK293T cells were transfected with pNSF-FBXO38 and lysed using an isotonic buffer containing Benzonase (0.125 U/μl). Lysates were precleared with 0.40 μm filters and Strep-tagged complexes were isolated using Strep-Tactin® Sepharose resins. In vitro ubiquitination assays were performed as described previously (Horn et al., 2014 (link)). Briefly, resins with affinity-purified FBXO38 were mixed with 30 μl of ubiquitination assay buffer containing 0.1 μM UBE1, 10 ng/ml Ubch3, 10 ng/ml Ubch5c, 1 μM ubiquitin aldehyde (all Boston Biochem), 2.5 μg/μl ubiquitin (Sigma Aldrich), 50 mM Tris (pH 7.6), ±2 mM ATP, 5 mM MgCl2, 0.6 mM DTT for 45 min at 37°C. Complexes were eluted by competition with 3.5 μl desthiobiotin containing Buffer E (IBA Lifesciences).
9
Antibody-based Western Blot Analysis Protocol
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Antibodies used for Western blot analysis included the following: anti-C1orf55/SDE2 (epitope: a.a. 318–410; Sigma-Aldrich), anti-GFP (JL-8, Clontech), anti-HA (6E2, Cell Signaling), anti-pCHK1 (S317, Cell Signaling), anti-Actin (Cell Signaling), anti-CDT1 (Cell Signaling), anti-Flag (M2, Sigma-Aldrich), anti-Tubulin (Sigma-Aldrich), Cyclin E (H-12, Santa Cruz), anti-PCNA (PC-10, Santa Cruz), anti-Vinculin (H-300, Santa Cruz), anti-GST (B-14, Santa Cruz), Cyclin A (H-432, Santa Cruz), anti-γH2AX (JBW301, Millipore), anti-CDT2 (Bethyl), anti-pRPA (S33, Bethyl), pKAP-1 (S824, Bethyl), anti-ORC2 (BD Pharmingen), and anti-MUS81 (MTA30 2G10/3, Abcam). Mitomycin C, camptothecin, hydroxyurea, cycloheximide, aphidicolin, and Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma-Aldrich. Rucaparib (AG-014699) was purchased from Selleckchem. MLN4924, ubiquitin vinyl sulfone, and ubiquitin aldehyde were purchased from Boston Biochem. Drugs were used at the concentrations indicated in the figure legends.
10
In Vitro and In Vivo CDK1 Ubiquitination
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The in vitro ubiquitination of CDK1 was performed in a volume of 10μl containing 50mM Tris-HCl (pH 7.6), 5mM MgCl2, 0.6mM DTT, 2mM ATP, 2μl in vitro transcribed/translated unlabeled F-box protein, 1.5ng/μl E1 (His6-ubiquitin activating enzyme, Boston Biochem), 10ng/μl His6-UbcH3 (E2, Boston Biochem), 10ng/μl UbcH5a (E2, Boston Biochem), 2.5μg/μl ubiquitin (Sigma), 1μM ubiquitin aldehyde (Boston Biochem), and 1μl 35S-methionine-labelled in vitro transcribed/translated CDK1 as substrate. The reactions were incubated at 30ºC for 1h and analyzed by SDS-PAGE and autoradiography.
The in vivo ubiquitination experiments were performed in Cos-7 cells transfected and treated with ConA 4 hours before harvesting. Cells were washed in PBS, lysed at 95ºC for 15 minutes in NP40 buffer supplemented with 5% SDS and 10mM iodoacetamide, and diluted in NP40 buffer supplemented with 10mM iodoacetamide. Flag-CDK1 was immunoprecipitated and proteins separated by SDS-PAGE, electroblotted and probed with different antibodies.
The in vivo ubiquitination experiments were performed in Cos-7 cells transfected and treated with ConA 4 hours before harvesting. Cells were washed in PBS, lysed at 95ºC for 15 minutes in NP40 buffer supplemented with 5% SDS and 10mM iodoacetamide, and diluted in NP40 buffer supplemented with 10mM iodoacetamide. Flag-CDK1 was immunoprecipitated and proteins separated by SDS-PAGE, electroblotted and probed with different antibodies.
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