20s proteasome

Manufactured by Enzo Life Sciences

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The 20S proteasome is a key enzymatic complex involved in the degradation of proteins within eukaryotic cells. It functions as the proteolytic core of the larger 26S proteasome, which is responsible for the majority of protein turnover and degradation in the cell.

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12 protocols using 20s proteasome

Human Erythrocyte 20S Proteasome Protocol

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20S proteasome, isolated from human
erythrocytes, was purchased from Enzo Life Sciences Inc. (Farmingdale,
NY). All reagents used in tests with proteasome were of molecular
biology grade. The pH of all buffers was determined at 20 °C.

Giżyńska M., Witkowska J., Karpowicz P., Rostankowski R., Chocron E.S., Pickering A.M., Osmulski P., Gaczynska M, & Jankowska E. (2018). Proline- and Arginine-Rich Peptides as Flexible Allosteric Modulators of Human Proteasome Activity. Journal of Medicinal Chemistry, 62(1), 359-370.

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Proteasome-mediated Degradation of Kinases

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200 ng of recombinant HRI (SignalChem) or 500 ng of recombinant eIF2α (abcam) were incubated with 2 µg of purified 20S proteasome (Enzo) in 20 mM Hepes pH 7.4/2 mM EDTA/1 mM EGTA for the indicated times at 37°C. When indicated, the 20S proteasome was incubated with 10uM MG132 for 30 min prior to its incubation with the kinase. Protein mixtures were loaded onto SDS-PAGE gels and stained o.n. with SYPRO Ruby Protein Gel Stain as indicated by the vendor. Gels were imaged using Gel Doc XR (Bio-Rad). In the figure the relative degradation of HRI+20S to HRI+20S+PI is shown.

Alvarez-Castelao B., tom Dieck S., Fusco C.M., Donlin-Asp P., Perez J.D, & Schuman E.M. (2020). The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition. eLife, 9, e52714.

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Proteasomal Cleavage of APE1 Protein

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The cleavage reaction assay was performed using 125 ng of purified recombinant proteins and 500 ng of 20S proteasome (Enzo Life Sciences, Farmingdale, NY, USA) in 50 mM Tris-HCl pH 7.5, 25 mM KCl, 10 mM NaCl, 1 mM MgCl₂ and 1 mM DTT, at 37 °C (final reaction volume 20 μL). After 1 h of incubation, the reaction was blocked with 4× Laemmli sample buffer. The reaction products were then resolved by SDS-PAGE, and immunoblotted for APE1.

Lirussi L., Antoniali G., Scognamiglio P.L., Marasco D., Dalla E., D’Ambrosio C., Arena S., Scaloni A, & Tell G. (2020). Cleavage of the APE1 N-Terminal Domain in Acute Myeloid Leukemia Cells Is Associated with Proteasomal Activity. Biomolecules, 10(4), 531.

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Proteasome Peptidase Activity Assay

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Trypsin-, chymotrypsin-, and caspase-like peptidase activities associated with the 20S proteasome were measured using the Proteasome-Glo™ Assay System (Promega, Madison, WI, USA). A 1 μg/ml of purified 20S proteasome (Enzo Life Sciences, Farmingdale, NY, USA) was incubated at room temperature for 30 min with vehicle, 20 μg/ml AMWAP or 1 μM MG-132 peptidase inhibitor positive control in a 96-well plate containing the substrate for one specific peptidase. Luminescence was then measured with an Infinite F200 Pro plate reader (Tecan, Crailsheim, Germany). A blank reaction without 20S proteasome was used to determine background luminescence associated with the vehicle and Proteasome-Glo™ reagent. The values of the blank reactions were subtracted from all experimental values. Relative luciferase units (RLUs) correspond to the levels of peptidase activity.

Aslanidis A., Karlstetter M., Scholz R., Fauser S., Neumann H., Fried C., Pietsch M, & Langmann T. (2015). Activated microglia/macrophage whey acidic protein (AMWAP) inhibits NFκB signaling and induces a neuroprotective phenotype in microglia. Journal of Neuroinflammation, 12, 77.

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Proteasomal Activity in Hypoxic Lung

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Chymotrypsin-like, trypsin-like, and caspase-like proteasome activities were
measured in mouse lung and mouse pulmonary artery after exposure to 3 weeks of
normoxia or hypoxia. Lung tissue from a single animal, or pulmonary arteries
pooled from four animals within the same treatment group, were prepared in
homogenization buffer (25 mM Tris pH 7.5, 100 mM NaCl, 5 mM ATP, 0.2% glycerol).
Samples were then centrifuged for 10 min at 5000 rpm at 4 ℃, the supernatant was
collected, and protein concentrations were determined by bicinchoninic acid
(BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL). To measure
proteasomal activity, samples were then incubated for 1 hour with assay buffer
[50 mM Tris pH 7.5, 2 mM dithiothreitol (DTT), 5 mM MgCl₂, 2 mM ATP]
containing the fluorogenic substrate [40 µM Suc-Leu-Leu-Val-Tyr-
7-amino-4-methylcoumarin (AMC), chymotrypsin-like; Boc-Leu-Arg-Arg-AMC,
trypsin-like; or Z-Leu-Leu-Glu-AMC, caspase-like; Enzo Life Sciences,
Farmingdale, NY]. Samples treated with either 1 µg purified human 20S Proteasome
(Enzo Life Sciences) or 40 nM MG132 were used as positive and negative controls,
respectively. Proteolytic activities were measured by observing the fluorescence
created by release of the AMC fluorescent group with a Wallac Victor 3
fluorimeter (PerkinElmer, Waltham, MA) every 10 minutes over 1 hour
(excitation = 390 nm and emission = 460 nm).

Wade B.E., Zhao J., Ma J., Hart C.M, & Sutliff R.L. (2018). Hypoxia-induced alterations in the lung ubiquitin proteasome system during pulmonary hypertension pathogenesis. Pulmonary Circulation, 8(3), 2045894018788267.

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Protein Aggregation and Ubiquitination Analysis

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All cell culture and standard reagents were purchased from Sigma. The region of bovine opsin used as an N-glycosylation reporter and epitope tag was as described previously (Johnson et al., 2012 (link)). Anti-TRC40 serum was a gift from Bernhard Dobberstein (ZMBH, Heidelberg, Germany). Commercially available antibodies against the following targets were purchased from the indicated suppliers: BAG6, BAP31, GFP, tubulin and V5 (Abcam), FLAG M2 and calnexin (Sigma), HSP70 (Stressgen), ubiquitin FK2, p23, 20S proteasome (Enzo Life Sciences) and LAMP1 (DHSB, University of Iowa). The antibody against GRASP65 was a gift from Martin Lowe (University of Manchester, UK). ProteoStat® reagent for the detection of protein aggregates was from Enzo. Bortezomib was from Selleck Chemicals, leupeptin and pepstatin A were from BIOMOL. RFP in pcDNA3.1⁺ was a gift from Viki Allan (Manchester, UK). siRNA duplexes for knockdowns were from Qiagen, and they targeted sequences that were identified previously (Winnefeld et al., 2006 (link)): SGTA target sequence, 5′-TTTGAAGCTGCCGTGCATT-3′; BAG6 target sequence, 5′-CAGCTCCGGTCTGATATACAA-3′.

Wunderley L., Leznicki P., Payapilly A, & High S. (2014). SGTA regulates the cytosolic quality control of hydrophobic substrates. Journal of Cell Science, 127(21), 4728-4739.

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Proteasomal Degradation of SUMOylated and Ubiquitinated CP2c

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In vitro SUMOylation and in vitro ubiquitination of bead-bound GST-CP2c were carried out with a SUMOylation kit (Enzo Life Sciences, BML-UW8955-0001) or Ubiquitination kit (Enzo Life Sciences, BML-UW9920-0001), respectively, according to the manufacturer’s procedures. Following 1-hour incubation at 37°C, the reaction was terminated by GST pull down. The modified GST-CP2c samples were incubated with 20S proteasome (Enzo Life Sciences, BML-AK740-0001) or 26S proteasome (Enzo Life Sciences, BML-PW8950-0001) in each assay buffer. After 1 hour of incubation at 37°C, the reaction was terminated with an SDS loading buffer. The samples prepared above were analyzed using Western blot.

Son S.H., Kim M.Y., Lim Y.S., Jin H.C., Shin J.H., Yi J.K., Choi S., Park M.A., Chae J.H., Kang H.C., Lee Y.J., Uversky V.N, & Kim C.G. (2023). SUMOylation-mediated PSME3-20S proteasomal degradation of transcription factor CP2c is crucial for cell cycle progression. Science Advances, 9(4), eadd4969.

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Assaying Proteasome Activities

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Proteasome activities were determined by the Proteasome-Glo Assay System (Promega) using purified human erythrocyte-derived 20S proteasome (Enzo Life Sciences).

Momose I., Abe H., Watanabe T., Ohba S.I., Yamazaki K., Dan S., Yamori T., Masuda T, & Nomoto A. (2014). Antitumor effects of tyropeptin-boronic acid derivatives: New proteasome inhibitors. Cancer Science, 105(12), 1609-1615.

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Proteasomal Degradation of S100-β Protein

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Purified human S100-β (23 (link)) was incubated with purified 20S proteasome (Enzo Life Sciences, Farmingdale, NY, USA) (molar ratio 250:1) in digestion buffer [30 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2 mM MgCl₂, 1 mM DTT, 0.01% sodium dodecyl sulfate) for 16 h at 37°C. As a control, the same digestion was set up with either no proteasome or with acetic acid (1%)–inactivated proteasome. Generated peptides were purified and concentrated using a cationic resin (ZipTip with strong cation exchange; MilliporeSigma) following the manufacturer’s instructions. Retained peptides were eluted from the resin and analyzed by MS.
The S100-β human protein sequence was analyzed in silico for potential proteasome and immunoproteasome cleavage sites using several published algorithms (26 (link), 27 (link)).

Calviño-Sampedro C., Gomez-Tourino I., Cordero O.J., Reche P.A., Gómez-Perosanz M., Sánchez-Trincado J.L., Rodríguez M.Á., Sueiro A.M., Viñuela J.E, & Calviño R.V. (2019). Naturally presented HLA class I–restricted epitopes from the neurotrophic factor S100-β are targets of the autoimmune response in type 1 diabetes. The FASEB Journal, 33(5), 6390-6401.

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Quantifying α-Synuclein Proteasome Degradation

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200 ng of purified α-synuclein (purchased from Novus Biologics) was incubated with 3.5 nM of purified 20S proteasome (purchased from Enzo Lifesciences) in 40 μL of 50 mM Tris-HCl (pH = 7.8). The agonist or DMSO was then added to the mixture and allowed to incubate for two hours at 37°C. The reaction was then quenched with SDS loading buffer (4×). All of the sample was loaded into a 4–20% SDS-PAGE gel. Quantitation was done by also loading 200 ng, 100 ng, 50 ng, and 25 ng of pure α-synuclein in the same gel to ensure no difference in quantitation was due to image contrast differences. After SDS-PAGE electrophoresis, the proteins were transferred to a nitrocellulose blot. The blot was then blocked with 5% skim milk at room temperature for one hour. The blot was then drained and 1:1000 of anti-α-synuclein antibody was added in 5% milk at 4°C overnight. Next the blot was rinsed with PBS-T (0.01% tween 20) 3 × 10 minutes. The secondary LI-COR antibody (anti-rabbit) was added at a dilution of 1:10,000 in LI-COR block for one hour at room temperature. After rinsing again 3 × with PBS-T the blot was scanned with a LI-COR Odyssey. All quantitation of gel bands was done with ImageJ.

Trader D.J., Simanski S., Dickson P, & Kodadek T. (2017). Establishment of A Suite of Assays That Support the Discovery of Proteasome Stimulators. Biochimica et biophysica acta, 1861(4), 892-899.

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