Microsomal membrane fractions were prepared from transiently transformed N. benthamiana leaves. Tissue was homogenized with 3 mL homogenization buffer per g fresh weight ( 50mM Hepes (pH 7.8), 500 mM sucrose, 1 % (w/v) PVP-40, 3 mM DTT, 3 mM EDTA, supplemented with Complete Protease Inhibitor Mixture (Roche) and Phosphatase Inhibitor Mix 1 (Serva)). The homogenate was centrifuged at 10,000 g for 20 min at 4 °C. The supernatant was filtered through MiraCloth and subsequently centrifuged at 100,000 g for 45 min at 4 °C. The microsomal pellet was resuspended in 5 mM Tris/MES (pH 6.5), 330 mM sucrose, 2 mM DTT, supplemented with Complete Protease Inhibitor Mixture (Roche) and Phosphatase Inhibitor Mix 1 (Serva).
Complete protease inhibitor mixture
Manufactured by Roche
Sourced in Germany, United States, Switzerland, China, France, Italy
Complete protease inhibitor mixture is a laboratory product that functions to inhibit the activity of proteases, which are enzymes that break down proteins. It is designed to be used in various research applications where the preservation of protein structure and function is critical.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Ripa buffer, by Merck Group (7 mentions)
Bradford assay, by Bio-Rad (7 mentions)
Phosstop, by Roche (6 mentions)
β actin, by Merck Group (6 mentions)
Rotiszint eco plus, by Carl Roth (5 mentions)
Bovine serum albumin (bsa), by Roche (5 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Diax 900 homogenizer, by Merck Group (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti actin, by Merck Group (5 mentions)
Cell culture media, by Thermo Fisher Scientific (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Las 3000 imaging system, by Fujifilm (4 mentions)
Ecl plu, by Cytiva (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Laemmli sample buffer, by Bio-Rad (4 mentions)
258 protocols using complete protease inhibitor mixture
1
Isolation of Microsomal Membranes from N. benthamiana
2
Subcellular Fractionation of Striatum and Midbrain
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Striatum and ventral midbrain from injected and noninjected hemispheres were rapidly dissected and homogenized with six volumes of Triton lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 1% Triton X-100, 1 mM EDTA, 1× EDTA-free Complete protease inhibitor mixture [Roche] and 1× phosphatase inhibitor mixture 2 and 3 [Sigma]). Tissues were disrupted using a mechanical homogenizer (IKA T10 basic, Ultra Turrax). The Triton-soluble fraction was obtained after ultracentrifugation at 100,000 × g for 30 min at 4 °C. The resulting pellets were further extracted by sonication in 3× volumes of SDS lysis buffer (50 mM Tris⋅HCl pH 7.4, 2% SDS, 1× EDTA-free Complete protease inhibitor mixture [Roche] and 1× phosphatase inhibitor mixture 2 and 3 [Sigma]). Samples were centrifuged at 21,000 × g for 30 min at 25 °C to obtain the Triton-insoluble (SDS-soluble) fraction. Protein concentration was determined using the BCA assay (Pierce Biotech).
3
Isolation of Microsomal Membranes from N. benthamiana
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Microsomal membrane fractions were prepared from transiently transformed N. benthamiana leaves. Tissue was homogenized with 3 mL homogenization buffer per g fresh weight ( 50mM Hepes (pH 7.8), 500 mM sucrose, 1 % (w/v) PVP-40, 3 mM DTT, 3 mM EDTA, supplemented with Complete Protease Inhibitor Mixture (Roche) and Phosphatase Inhibitor Mix 1 (Serva)). The homogenate was centrifuged at 10,000 g for 20 min at 4 °C. The supernatant was filtered through MiraCloth and subsequently centrifuged at 100,000 g for 45 min at 4 °C. The microsomal pellet was resuspended in 5 mM Tris/MES (pH 6.5), 330 mM sucrose, 2 mM DTT, supplemented with Complete Protease Inhibitor Mixture (Roche) and Phosphatase Inhibitor Mix 1 (Serva).
4
Microsomal Membrane Fractionation from Transiently Transformed N. benthamiana
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Microsomal membrane fractions were prepared from transiently transformed N. benthamiana leaves. Tissue was homogenized with 3 ml homogenization buffer per gram fresh weight (50 mM Hepes pH 7.8, 500 mM sucrose, 1% PVP-40, 3 mM dithiothreitol (DTT), 3 mM EDTA, supplemented with Complete Protease Inhibitor Mixture (Roche, Basel, Switzerland) and Phosphatase Inhibitor Mix 1 (Serva, Heidelberg, Germany)). The homogenate was centrifuged at 10,000 × g for 20 min at 4 °C. The supernatant was filtered through MiraCloth and subsequently centrifuged at 100,000 × g for 45 min at 4 °C. The microsomal pellet was resuspended in 5 mM Tris/MES pH 6.5, 330 mM sucrose, 2 mM DTT, supplemented with Complete Protease Inhibitor Mixture (Roche) and Phosphatase Inhibitor Mix 1 (Serva).
5
Membrane Protein Isolation from E. coli
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E. coli strains producing recombinant proteins were harvested by centrifugation (2000 × g). The pellet of 250 A600 units equivalent of bacterial cells was resuspended in 1.5 ml of buffer A (10 mm Tris, pH 7.6, 10 μg/ml DNase (Sigma), Complete Protease Inhibitor Mixture (Roche Applied Science), and sucrose 20% (w/w) (Sigma)). The cells were passed through a French press cell disruptor (SIM-AMICO) at 1500 pressure units (p.s.i.) using a 3/8-inch-diameter piston (20K French pressure cell, Thermo). Unbroken cells and possible inclusion bodies were removed by two cycles of centrifugation at 4 °C for 15 min at 1600 × g. The resulting supernatant was centrifuged at 4 °C for 1 h at 125,000 × g. The collected crude membrane pellet was resuspended in 0.5 ml of buffer M (10 mm Tris, pH 7.4, Complete Protease Inhibitor Mixture (Roche Applied Science), and sucrose 20% (w/w)). The membrane fraction was then added on top of a discontinuous sucrose gradient consisting of 1.5-ml layers ranging from 60% (bottom) to 30% by 5% increments and separated according to the method described previously (41 (link)). Fractions of 500 μl were collected, and the protein content was analyzed by SDS-PAGE and Western blot.
6
Cell Lysis Protocol for Adherent and Suspended Cells
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Cell lysates were prepared as described previously with minor modifications25 (link). In brief, confluent cells were washed with cold PBS and then scraped from plates in the presence of cold lysis buffer containing 20 mM Tris (pH 8.0), 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 0.5% sodium deoxycholate, and complete protease inhibitor mixture (Boehringer Mannheim). Floating cells in the conditioned media were collected via centrifugation and resuspended in cold lysis buffer. Cell lysates obtained from confluent and floating cells were centrifuged for 15 min at 4 °C to remove cell debris. The aliquots were stored at − 70 °C until further use.
7
Glutamate-Induced Signaling Pathway Regulation
8
Protein Extraction and Western Blot Analysis
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Cells were lysed for 15 min at 4 °C in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Nonidet P40) supplemented with protease inhibitors (CompleteTM protease inhibitor mixture, Roche, Rotkreuz, Switzerland). Supernatants were collected, and the protein concentration was determined with a BCA assay (Sigma, Deisenhofen, Germany). Equal amounts of protein samples were separated on 8% Tris/glycine gels and then transferred to nitrocellulose membranes (GE Healthcare, Uppsala, Sweden). Subsequently, the membranes were incubated with primary antibodies and HRP-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania, USA). Chemiluminescence was measured, using an imager and the software Fusion (Vilber Loumat).
9
Western Blot Analysis of Metabolic Proteins
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Tissue extracts were prepared with RIPA lysis buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with a PhosStop phosphatase inhibitor and cOmpleteTM protease inhibitor mixture (Roche). Protein extracts were resolved by SDS PAGE. Blots were incubated overnight at 4°C with antibodies against the following proteins (source, catalog number, and dilution of the antibody are given in parentheses): IDE (Origene, TA327113, 1:1000), LIPIN1 (Cell Signaling, #14906, 1:1000), IRE1α (Cell Signaling, #3294, 1:1000) , phospho-IRE1α Ser724 (Novus Biologicals, NB100-2323, 1:1000 ), AKT (Cell Signaling, #4691, 1:2000), phosphor-AKT Ser473 (Cell Signaling, #4060, 1:1000), GSK3β (Cell Signaling, #12456, 1:2000), phospho-GSK3β Ser9 (Cell Signaling, #5558, 1:1000), GAPDH (Santa Cruz, sc-32233, 1:2000). A polyclonal rabbit anti-human TM6SF2 antibody was raised against a peptide corresponding to the C-terminal 15 amino acids of human TM6SF2 CPPPSDPLALHKKQH (YenZym Antibodies, LLC, CA). After washing, membranes were incubated with an IRDye-conjugated IgG (LI-COR Biosciences, Lincoln, NE) secondary antibody diluted 1:5000 for 1 h. The intensity of the protein bands was quantified using an image processing program (LI-COR Biosciences, Lincoln, NE).
10
Protein Extraction from ASML Cell Line
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Cell pellets from the ASML cell line were collected and re-suspended in buffer containing 0.1 M NaCl, 10 mM Tris-HCl and 1 mM ethylenediaminetetraacetic acid (EDTA). Thereafter, an equal quantity of lysis buffer (100 mM Tris-HCl, 4% sodium dodecyl sulfate (SDS), 20% glycerol) was added. Both buffers were supplemented with Complete TM protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany) as recommended by the manufacturer. The lysis buffer was additionally supplemented with dithiotreitol (DTT) to a final concentration of 200 mM. Lysates were boiled for 10 min at 99°C in a Thermomixer (Eppendorf, Hamburg, Germany) at a mixing frequency of 500 rpm and thereafter centrifuged at 10,600 × g for 10 min at 4°C. The protein concentration in a portion of each sample, lysed without DTT, was determined using the Pierce BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA) according to the recommendations of the manufacturer.
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