Sequential fractionation using RAB, RIPA, 70% formic acid (FA) buffer was performed as described previously36 (link) with modifications. Briefly, cortices from left hemisphere were weighted and homogenized using a dounce homogenizer (DWK Life Science #357422) for 25 strokes in 10-fold volume of ice-cold RAB buffer (100 mM MES, 1 mM EGTA, 0.5 mM MgSO4, 750 mM NaCl, 20 mM NaF, 1 mM Na3VO4, pH 7.0, supplemented with PhosSTOP, cOmplete-mini (Roche)). After ultracentrifugation for 20 min at 50,000 × g at 4 °C, the supernatant was collected and saved as the RAB-soluble fraction and the pellet was resuspended in 10-fold volume of ice-cold RIRA buffer (25 mM Tris, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 20 mM NaF, 1 mM Na3VO4, pH 8.0, supplemented with PhosSTOP, cOmplete-mini (Roche)) and nutated for 30 min at 4 °C. After ultracentrifugation for 20 min at 50,000 × g at 4 °C, the supernatant was collected and saved as the RIPA-soluble fraction and the pellet was resuspended in 3-fold volume of ice-cold 70% FA and nutated for 30 min at 4 °C. After ultracentrifugation for 20 min at 50,000 × g at 4 °C, the supernatant was collected and saved as the FA-soluble fraction. All fractions were stored at –80 °C.
Complete mini
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The Complete Mini is a compact, automated system designed for sample preparation and analysis. It provides reliable and consistent results for a variety of applications. The device offers an efficient and streamlined workflow to support laboratory operations.
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Lab products found in correlation
Phosstop, by Roche (162 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (61 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (38 mentions)
Protein assay reagent, by Bio-Rad (35 mentions)
Ripa buffer, by Merck Group (34 mentions)
Pvdf membrane, by Merck Group (27 mentions)
β actin, by Merck Group (25 mentions)
Du 800 spectrophotometer, by Bio-Rad (24 mentions)
Bradford assay, by Bio-Rad (23 mentions)
Protein assay, by Bio-Rad (22 mentions)
Ripa buffer, by Thermo Fisher Scientific (22 mentions)
Protein a sepharose beads, by GE Healthcare (18 mentions)
Cell lysis buffer, by Cell Signaling Technology (16 mentions)
Pvdf membrane, by Bio-Rad (15 mentions)
Nitrocellulose membrane, by Bio-Rad (14 mentions)
Phosphostop, by Roche (14 mentions)
Immobilon p membrane, by Merck Group (14 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (13 mentions)
Bca assay, by Thermo Fisher Scientific (13 mentions)
Dc protein assay, by Bio-Rad (12 mentions)
1 265 protocols using complete mini
1
Protein Fractionation from Brain Cortex
2
Purification of IBDV and VP2 VLP
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IBDV was purified following established procedures [11 (link)]. Briefly, supernatants of IBDV-infected QM7 cells were harvested at 72 h post-infection (hpi) and precipitated with 3.5% polyethylene glycol (PEG) 6000, 0.5 M NaCl and incubated (overnight, 4 °C, with mild shaking). The precipitate was isolated by centrifugation (3000 × g, 30 min, 4 °C), resuspended in PES buffer (50 mM PIPES pH 6.2, 150 mM NaCl, 20 mM CaCl2) supplemented with protease inhibitors (Complete Mini, Roche), and further purified by ultracentrifugation through a 25% sucrose cushion (170 000 × g, 150 min) followed by a 25–50% linear sucrose gradient (200 000 × g, 45 min).
To produce HT-VP2-466 VLP, H5 cell monolayers were infected with rBV HT-VP2-466 at a multiplicity of infection (moi) of 5 plaque-forming units per cell (pfu/cell) and harvested at 48 hpi. The cell pellet was lysed in PES buffer supplemented with 1% Igepal CA-630 (Sigma) and protease inhibitors (Complete Mini, Roche) for 20 min. The cell suspension was clarified by centrifugation, the supernatant loaded on a 25% sucrose cushion and centrifuged (170 000 × g, 150 min). The pellet was resuspended in PES buffer and centrifuged in a 25–50% linear sucrose gradient (200 000 × g, 45 min). Fractions (1 ml) were concentrated 10-fold by ultracentrifugation (240 000 × g, 120 min). All purification steps were performed at 4 °C.
To produce HT-VP2-466 VLP, H5 cell monolayers were infected with rBV HT-VP2-466 at a multiplicity of infection (moi) of 5 plaque-forming units per cell (pfu/cell) and harvested at 48 hpi. The cell pellet was lysed in PES buffer supplemented with 1% Igepal CA-630 (Sigma) and protease inhibitors (Complete Mini, Roche) for 20 min. The cell suspension was clarified by centrifugation, the supernatant loaded on a 25% sucrose cushion and centrifuged (170 000 × g, 150 min). The pellet was resuspended in PES buffer and centrifuged in a 25–50% linear sucrose gradient (200 000 × g, 45 min). Fractions (1 ml) were concentrated 10-fold by ultracentrifugation (240 000 × g, 120 min). All purification steps were performed at 4 °C.
3
Isolation and Characterization of Extracellular Vesicles
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The U937 cell line was grown in RPMI 1640 media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/ml penicillin-streptomycin. The MCF7 cells were grown in DMEM media (Sigma-Aldrich) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin. The cell lines are commercially available from ATCC (U937 ATCC# CRL-1593.2 & MCF7 ATCC# HBT-22). MCF7 and U937 cells were grown to 75% confluence without FBS for 48 h prior to EV isolation. U937 cells were centrifuged 3 min at 1,200 × g to recover a cell free supernatant. Supernatants from cultures of both cell lines were used for isolation of EVs, and protease inhibitors were added (Complete Mini, Roche). The cell free supernatants were then sequentially centrifuged at 3,000 × g for 10 min and at 10,000 × g for 10 min, filtered through a 0.45 μm PES filter (VWR) and then finally centrifuged at 100,000 × g for 2 h, followed by one washing step and a second centrifugation at 100,000 × g for 2 h. Pelleted EVs were resuspended in a small amount of sterile filtered PBS supplemented with protease inhibitors (Complete Mini, Roche). Samples were stored at −80 °C and later prepared for further analysis.
4
Mouse Testis Lysate Preparation
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Mouse testis lysate was prepared for western blotting as previously described (Hwang et al., 2019 (link); Ded et al., 2020 (link)). In short, mouse testes were homogenized in 0.32M sucrose and centrifuged at 1000 g for 10 min at 4°C to remove cell debris and nuclei. 1% Triton X-100 in PBS containing protease inhibitor cocktail (cOmplete Mini, EDTA-free, Roche) was added to the cleared homogenates to make total testis lysate. The lysates were centrifuged at 4°C, 14,000 g for 30 min and the supernatant was used for the downstream experiments. For experiments measuring ABCA1 expression, testes were homogenized in 1x RIPA buffer containing protease inhibitor cocktail (cOmplete Mini, EDTA-free, Roche), centrifuged at 4°C, 14,000 g for 30 min and the supernatant was used for the downstream experiments.
5
Quantitative Western Blot Analysis of Metabolic Proteins
6
Purification of Bacterial Chemoreceptor Proteins
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Membrane fractions containing MCP proteins were prepared as described (Schmidt et al., 2011 (link); Xu et al., 2016 (link)). HCB721 is deficient in all E. coli MCPs and cytoplasmic chemotaxis proteins except for the CheZ phosphatase. Cells were grown at 37°C in 150 ml LB supplemented with 100 μg/ml ampicillin. MCP expression was induced with 1 mM IPTG at OD600 0.5–0.7 for 3 h at 30°C, cells were harvested by centrifugation and re-suspended in the buffer containing 100 mM potassium acetate, 50 mM HEPES, pH 7.5, 5 mM magnesium acetate, 0.05% (v/v) β-mercaptoethanol, protease inhibitors (Complete mini, EDTA free, Roche), and Benzonase Nuclease (Novagen). Cells were lysed by sonication and cell debris was removed by centrifugation for 20 min at 7,000 × g, the supernatant was loaded on a sucrose step gradient (0.5, 1.5, and 2 M) and centrifuged at 100,000 × g for 1 h at 4°C. The second band was removed, diluted with water containing protease inhibitors (Complete mini, with EDTA, Roche) and centrifuged for 1 h at 100,000 × g at 4°C. The pellets were resuspended in a small amount of storage buffer (50 mM NaH2PO4, 1 mM EDTA, 10% (v/v) glycerol). The membrane fraction samples were stored as single-use aliquots at 70°C.
7
Nuclear Fractionation of S2 Cells
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S2 cells overexpressing FLAG-ouib or FLAG-M1BP were collected and washed with TBS. Cells were then centrifuged at 4000 g at 4°C for 5 min. The pellet was suspended and vortexed with 400 μl Buffer A [10 mM Hepes pH 7.9, 10 mM KCl, 1 mM DTT and 1 unit Complete Mini (Roche)] and 25 μl 10% NP-40. Then sample was centrifuged at 1500 g at 4°C for 5 min. The pellet was suspended and vortexed with 50 μl Buffer C [20 mM Hepes pH7.9, 400 mM NaCl, 2 mM MgSO4, 1mM DTT and Complete Mini (Roche)], then shaked at 4°C for 30 min. After shaking, sample was centrifuged at 14,000 rpm at 4°C for 5 min and supernatant was collected.
8
Membrane Fraction Isolation and Trpm5 Detection
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Tissues were collected from spinal cord lumbar enlargements and frozen after removing the dorsal and ventral roots. For the membrane fraction, corresponding to the plasma membrane-enriched fraction, samples were homogenized in ice-cold lysis buffer (320 mM sucrose, 5 mM Tris-HCL pH 7.5, 10 µM iodoacetamide) supplemented with protease inhibitors (CompleteMini, Roche diagnostic Basel, Switzerland). Unsolubilized material was pelleted by centrifugation at 7000 × g for 5 min. The supernatant was subjected to an additional centrifugation step at 18,000 × g for 70 min at 4 °C. Pellets were collected and homogenized in ice-cold lysis buffer (1% Igepal CA-630, PBS 1×, 0.1% SDS, 10 µM iodoacetamide), supplemented with protease inhibitors (CompleteMini, Roche diagnostic). Protein concentrations were determined using a detergent-compatible protein assay (Bio-Rad). Equal protein amounts (30 µg) from samples were size fractionated by 4–15% Mini-PROTEAN TGX stain-free gels (Bio-Rad), transferred to a nitrocellulose membrane and probed with a polyclonal Trpm5 antibody (1:300, ACC-045, Alomone from rabbit) at 4 °C overnight in Tris-buffered saline containing 5% fat-free milk powder. The blot was then incubated for 1 h at 22 °C with a polyclonal horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:40,000; AB228341, ThermoFisher).
9
Nuclear Fractionation of B Cells
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Protein extract preparation and Western blot analysis were performed as previously described (27 (link), 31 (link)). Antibodies are listed in Supplementary Table 3
10
Evaluating Caspase-9 and P53 Expression in Chemotherapy
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Western blot enabled the determination of the effect of the combination of AA with each chemotherapeutic drug on the expression of caspase-9 and P53. Total protein extracts were prepared using RIPA buffer supplemented with completeTM Mini (Roche) and protein concentration was determined by BCATM protein assay (Pierce). Western blot analyses were done according to a previously described protocol (Pires et al., 2016 (link)). The primary antibodies anti-caspase-9 (Santa Cruz Biotechnology Cat# sc-8355, RRID:AB_2071323 ), anti-P53 (Santa Cruz Biotechnology Cat# sc-47698, RRID:AB_628083 ) and anti-actin (Sigma, A5141) and the secondary antibodies goat-anti-mouse (GE Healthcare) and goat anti-rabbit (Santa Cruz Biotechnology Cat# sc-2007, RRID:AB_631740 ) were used. Standard protein markers (Nzytech, MB09002) were also used. Membranes were scanned using Typhoon FLA 9000 equipment.
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