Tissue lysates were prepared as described previously with some modifications (Mao et al., 2016 ). Nonionic detergent-soluble and -insoluble fractions were prepared by sequential lysis buffer. Tissues were homogenized in the following lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, phosphatase inhibitor cocktail II and III (Sigma-Aldrich), and complete protease inhibitor mixture (Sigma-Aldrich)], and then were centrifuged and soluble supernatant was collected. The insoluble pellet was washed once in brain lysis buffer containing nonionic detergent (0.1% Triton X-100) and the resulting pellet was lysed with lysis buffer containing 2% SDS and 0.5% sodium deoxycholate. The homogenate was centrifuged and the resulting supernatant (Triton X-100 detergent-insoluble) was collected. Total lysates were prepared by homogenization of tissue in RIPA buffer [50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1% SDS, 0.5% sodium-deoxycholate, phosphatase inhibitor cocktail II and III (Sigma-Aldrich), and complete protease inhibitor mixture (Sigma-Aldrich)]. After homogenization, samples were rotated at 4 °C for 30 min for complete lysis, the homogenate was centrifuged at 22,000 × g for 20 min and the supernatants were collected. Protein levels were quantified using the BCA Kit (Pierce, Rockford, IL, USA) with BSA standards and analyzed by immunoblot.
Complete protease inhibitor mixture
Manufactured by Merck Group
Sourced in United States, China
The Complete Protease Inhibitor Mixture is a laboratory product designed to inhibit a broad range of proteases. It is a premixed solution containing various protease inhibitors that can be used to prevent protein degradation during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Anti actin, by Merck Group (3 mentions)
Anti sirt7, by Santa Cruz Biotechnology (2 mentions)
Anti ddb1, by Merck Group (2 mentions)
Anti flag, by Abnova (2 mentions)
Anti dcaf1, by Santa Cruz Biotechnology (2 mentions)
Anti flag magnetic beads, by Merck Group (2 mentions)
Anti ha, by Fortrea (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail 2 and 3, by Merck Group (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Hrp conjugated secondary ab, by GE Healthcare (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Caspase 3, by Enzo Life Sciences (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs hq, by Bio-Rad (1 mentions)
Mg132, by Avantor (1 mentions)
Protein g agarose, by Cell Signaling Technology (1 mentions)
27 protocols using complete protease inhibitor mixture
1
Tissue Fractionation and Protein Quantification
2
Whole Cell Extract and Nuclear Protein Isolation
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To obtain whole cell extract, SKO-007(J3) cells were lysed for 30 min on ice in lysis buffer [1% Nonidet P-40 (v/v), 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM phenylmethyl-sulfonyl fluoride, 10 mM NaF, 1 mM Na3VO4, complete protease inhibitor mixture (Sigma Aldrich) in PBS]. Nuclear extracts were prepared from SKO-007(J3) cells as previously described [56 (link),57 (link)]. Protein concentration was determined by the BCA method (ThermoFisher Scientfic, Waltham, MA, USA). Thirty to 50 μg was resolved by SDSPAGE and transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany). After blocking in milk, membranes were probed with specific Abs. Antibodies against p65 and Oct-1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA)). Antibody against β-actin was purchased from Sigma Aldrich. An HRP-conjugated secondary Ab and an ECL detection system (Amersham, GE Healthcare, London, United Kingdom) were used to reveal immunoreactivity.
3
Quantification of Apoptosis-Related Proteins
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Cell lines were lysed in RIPA buffer (100 mM Tris–HCL pH 8, 150 mM NaCl, 1 % Triton X-100, 1 mM MgCl, 25 mM NaVO4) in the presence of complete protease-inhibitor mixture (Sigma). Immunoblotting was performed with antibodies to: caspase-9 (rabbit polyclonal antibody, Enzo Life Sciences); caspase-8 (mouse monoclonal antibody, Transduction Laboratories); caspase-3 (mouse monoclonal antibody, Enzo Life Sciences); cathepsin B (rabbit polyclonal antibody, Calbiochem). As a control, the membranes were incubated with specific antibodies of anti-α-tubulin (mouse monoclonal antibody, Sigma). The intensities of bands of active fragments were quantified normalizing to Tubulin. The optical density of the bands [integrated area in arbitrary units (AU)] was measured by using the National Institutes of Health Image J software (rsb.info.nih.gov/ij ).
4
Isolation and Analysis of HCAEC Proteins
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Proteins were isolated from HCAEC using a cell lysis buffer consisting of 2.5 mM EDTA, 20 mM Tris pH 7.4, 100 mM NaCl, 1 mM Na3VO4, 1% Triton X-100, 10 mM NaF, 1% sodium deoxycholate, 0.1% SDS, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, and 1 tablet/10 ml EDTA-free complete protease inhibitor mixture (Sigma, St Louis, MO). Whole-cell extracts prepared from HCAEC were resolved in 4–20% bis-Tris polyacrylamide gels (Thermoscientific, Walthem, MA), followed by transfer to nitrocellulose membranes. Membranes were probed with anti-p65 antibody at 1∶2000 dilution in TBST buffer (Tris-Buffered Saline and Tween 20). Proteins were visualized by incubation with peroxidase-coupled secondary Abs in the presence of LumiGlo reagent while exposing in a Bio-Rad Chemidoc XRS/HQ.
5
Co-immunoprecipitation of proteins in HEK293T cells
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Human embryonic kidney cell lines (HEK293T from ATCC) were maintained in advanced DMEM, supplemented with Glutamine and 10% (v/v) fetal bovine serum. A day before transfection, approximately 3 × 106 cells were seeded on a 100 mm dish. Transfection was performed with a mixture of pcDNA plasmids expressing specific proteins, as indicated, using Lipofectamine 3000 (Thermo Fisher Scientific) with empty pcDNA vector plasmid as a balance. After 48 h, cells were harvested and lysed with sonication in the lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, pH 7.5) with complete protease inhibitor mixture (Sigma). Some cell lysate was mixed with loading buffer and heated at 95 °C for 5 min. For immunoprecipitation, the cleared cell lysate was incubated with 20 µL of anti-FLAG magnetic beads (Sigma) with shaking at 4 °C for 5 h. The beads were washed with the lysis buffer three times and bound proteins were eluted with FLAG peptides at 0.1 mg/mL. Samples were subjected to SDS-PAGE and Western Blotting analysis with appropriate antibodies. Antibodies used in the currernt study included anti-HA (COVANCE), anti-FLAG (Abnova), anti-DDB1 (Sigma), anti-Actin (Sigma), anti-DCAF1 (Santa Cruz), anti-SIRT7 (Santa Cruz), anti-Goat IgG (Santa Cruz), anti-Rabbit IgG (Sigma), and anti-Mouse IgG (Sigma).
6
Immunoprecipitation of Protein Complexes
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For each immunoprecipitation, 12 × 106 cells were treated for 24 h with OHT to activate CRE–ER and 1.5 h with 25 µM MG132 (VWR). Cell lysates were prepared in Kischkel buffer (50 mM Tris⋅HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and Complete Protease Inhibitor mixture [Sigma-Aldrich]), briefly sonicated, and subject to immunoprecipitation overnight with the 12CA5 antibody. Immune complexes were captured with Protein-G agarose (Cell Signaling Technologies), washed in Kischkel buffer, and resolved by SDS/PAGE followed by immunoblotting.
7
Western Blot Analysis of Cellular Proteins
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Cells were collected and lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) in the presence of complete protease-inhibitor mixture (Sigma-Aldrich). Protein concentration was determined by micro BCA assay (Pierce, Rockfod, IL, USA) and the indicated proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF, BioRad) or nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA). Membranes were probed using the following antibodies: anti-β-actin (1:10 000, Sigma-Aldrich); anti-calnexin (1:2000, Abcam, Cambridge, UK); anti-Tom20 (1:10000, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti- lactate dehydrogenase (LDH) (1:2000, Rockland); anti-long-chain fatty-acid CoA synthases (FACL4) (1:500, Santa Cruz Biotechnology); anti-calreticulin (CRT) (1:1000, Abcam); anti-c-FLIP Dave II clone (1:800, Alexis, Lausen, Switzerland); anti-RTN4 (1:1000, Abcam); anti-CLIMP63 (1:1000, Proteintech Group, Chicago, IL, USA); anti-cleaved caspase-3 (1:500, Cell Signaling Technology) and anti-PARP (1:1000; Cell Signaling Technology).
8
Protein Extraction and Western Blot Analysis
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24 h after transfection, cells were harvested and disrupted in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Nonidet P-40/0, 0.25% deoxycholate, 1 mM EDTA, and 50 mM Tris, pH 7.4) in the presence of complete protease inhibitor mixture (Sigma-Aldrich). 20 brains from larvae or 100 whole larvae for each genotype were used for protein extraction. Samples were rinsed in radioimmunoprecipitation assay buffer with complete protease inhibitor mixture (Roche) and 1 mM PMSF (Sigma-Aldrich), homogenized in a 1-ml glass/Teflon potter (Wheaton), and then spun at 7,000 g for 10 min. Supernatants were collected and boiled for 5 min in Laemmli buffer (4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol, and blue bromophenol). Extracted proteins were separated by 4–12% SDS-PAGE (NuPAGE; Invitrogen), transferred onto polyvinylidene difluoride (Bio-Rad Laboratories) membranes, and probed using the following antibodies: anti-Actin (1:2,000; EMD Millipore), anti-Myc (1:1,000 [BD] or 1:1,000 [Roche]), anti-V5 (1:1,000; Invitrogen), anti-Marf (1:500), anti-BiP/Grp78 (1:1,000; BD), and anti-BiP (1:500).
9
Nucleocytoplasmic Fractionation of HaCaT Cells
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The nucleocytoplasmic fractionation was performed using the CelLytic NuCLEAR extraction kit from Sigma. Briefly, HaCaT cells were stimulated as described in the figure legend and collected by trypsinization. Hypotonic lysis buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, and complete protease inhibitor mixture from Sigma-Aldrich) was added to the cells and incubated for 15 min on ice. Then cytoplasmic and nuclear fractions were isolated according to the protocol. For immunoprecipitations from nuclear fractions, the lysis buffer described above was used.
10
Isolation of Candida albicans Mitochondria
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Candida albicans cells were cultured overnight in the appropriate medium and harvested by centrifugation at 4,000 g for 10 min at 4°C, washed with 50 ml of ice‐cold water and resuspended in 0.6 M sorbitol, 50 mM Tris‐HCl pH 7.5 supplemented with complete protease inhibitor mixture (Sigma). Cells were disrupted by vortexing with 0.45 mm‐diameter sterile glass beads six times, for 30 s each, at 2‐min intervals, on ice. All subsequent steps were carried out at 4°C. Glass beads and unbroken cells were removed by centrifugation at 4,000 g for 10 min, and the supernatants were centrifuged again (14,000 g, 10 min, 4°C). The resulting pellets (corresponding to the mitochondria) were resuspended in 0.6 M sorbitol, 50 mM Tris‐HCl, pH 7.5, and the supernatants constituted the cytosolic fraction. Purified mitochondria were immediately frozen at −80°C.
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Candida albicans cells were cultured overnight in the appropriate medium and harvested by centrifugation at 4,000 g for 10 min at 4°C, washed with 50 ml of ice‐cold water and resuspended in 0.6 M sorbitol, 50 mM Tris‐HCl pH 7.5 supplemented with complete protease inhibitor mixture (Sigma). Cells were disrupted by vortexing with 0.45 mm‐diameter sterile glass beads six times, for 30 s each, at 2‐min intervals, on ice. All subsequent steps were carried out at 4°C. Glass beads and unbroken cells were removed by centrifugation at 4,000 g for 10 min, and the supernatants were centrifuged again (14,000 g, 10 min, 4°C). The resulting pellets (corresponding to the mitochondria) were resuspended in 0.6 M sorbitol, 50 mM Tris‐HCl, pH 7.5, and the supernatants constituted the cytosolic fraction. Purified mitochondria were immediately frozen at −80°C.
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