Proteasome assays were performed as described previously [68 (link)], using the Suc-LLVY-Luciferin substrate for chymotrypsin-like activity of the proteasome (the Proteasome Glo kit, Promega). In brief, cells were detached and washed in DMEM/10, followed by several washes in cold PBS. Proteasome lysis buffer (50 mM Tris-HCl, pH 7.5, 0.025% digitonin, 250 mM sucrose, 5 mM MgCl2, 0.5 mM EDTA, 2 mM ATP, and 1 mM DTT) was added to the cells and incubated on ice for 5–10 min. The lysates were then centrifuged for 15 min at 20,000 g to isolate the cytoplasm containing the proteasomes. The supernatant was transferred to a fresh tube, and equal amounts of protein were used in each assay.
Proteasome glo kit
Manufactured by Promega
The Proteasome-Glo kit is a luminescent assay designed to measure proteasome activity in cell lysates or purified proteasome samples. The kit uses a specific luminogenic substrate to detect the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities.
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Lab products found in correlation
7 protocols using proteasome glo kit
1
Proteasome Activity Measurement Protocol
2
Proteasomal Activity Measurement in HepG2 Cells
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Proteasomal activity was measured with the Proteasome-Glo kit (G8660, Promega). HepG2 cells were treated with siRNA twice as described above and plated equally (approximately 1 × 104 cells per well) in 96-well white-walled plates. The cells were cultured with or without 5 μM lactacystin for 2 hr before measurement. Proteasome-Glo Cell-Based Reagent was prepared as per manufacturer’s protocol and an equal volume was added to each well. The plate was mixed for 2 min using TAITEC E-36 micromixer and then incubated at room temperature for 10 min. Luminescence was measured by a microplate leader EnSpire (2300–00J, PerkinElmer).
3
Proteasomal activity in HIV-infected cells
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HPT1b cells were transduced with no virus, HR-IRES-EGFP (EGFP control), NL4-3ΔG/P-EGFP (HIV), or HR-VPR-IRES-EGFP (Vpr) as above. Three days after transduction, the trypsin-like, chymotrypsin-like, and caspase-like proteasomal activities were assayed using the Proteasome Glo kit (Promega) according to manufacturer’s instructions. Luminescence was detected using a Varioskan Lux multimode plate reader (Thermo Scientific).
4
Measuring Proteasomal Activity in Myoblasts
5
Proteasome Activity Assay in Insects
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Animals were collected 3 to 4 d after eclosion and frozen immediately on dry ice. Microcentrifuge tubes were weighed before and after on an analytical balance to determine the mass. Whole bodies were ground and incubated for 30 min on ice in 0.5 mL of lysis buffer (50 mM HEPES, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100). Lysates were then separated by centrifugation at 22000 g for 15 min at 4°C. In a 96-well black plate 50 μL of lysate was added to 50 μL of chymotrypsin-like, caspase-like, trypsin-like peptide substrates from the Proteasome-Glo kit (Promega, G8531). The substrates incubated with the lysates for 10 min before the luminescence reading was measured on a Synergy H4 Hybrid (BioTek, Winooski, VT USA). Relative luminescence was then determined proportionally to total mass per sample.
6
Viability and Apoptosis Assays for Cell Lines
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Viability of HEK293FT cells and primary neurons was analyzed according to the manufacturer’s instructions in 96 well plates: LDH Cytotox Non-Radioactive cytotoxicity assay (Promega), Caspase-glo 3/7 assay (Promega), TUNEL in situ cell death detection TMR red assay (Roche). For the TUNEL assay dead and living cells were counted manually with the Fiji cell counter plugin. At least 400 cells per condition were counted per experiment in a total of three independent experiments. Proteasome activity was measured using the Proteasome-Glo kit according to the manufacturer’s instructions (Promega).
7
Measuring Proteasomal Activity in Myoblasts
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Chymotrypsin-like proteasomal activity was measured with the Proteasome-Glo kit from Promega (G8660). Myoblasts were scraped from a T175 fiask, re-suspended in growth medium, and added to a 96-well white-walled plate at a seeding density of 10,000 cells/ well. At 80–90% confluence, cells were treated with LLC1 CM or non-CM with or without added 1α,25(OH)2D3 (10−8M) or ethanol for 24 h prior to the experiment. 100 μL of Proteasome-Glo cell-based reagent was added to 100uL of sample and incubated for 10 min. Luminescence was measured on a SpectroMax M2e.
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