Chinese hamster ovary cells were transfected with pEGFP‐N1 and plasmids encoding VipA‐EGFP variants using the jetPEI™ transfection reagent (Polyplus) according to manufacturer's protocol. Cells were harvested 24 h post transfection and co‐immunoprecipitations were performed using Chromotek (Hauppauge, New York, USA) GFP‐Trap agarose beads essentially as described by the manufacturer. In brief, cells were lysed with lysis Buffer (50 mmol/L Tris‐HCl pH 8, 150 mmol/L NaCl, 0.1 mmol/L EDTA, 0.5% [w/v] IGEPAL®, Company: Sigma‐Aldrich: St. Louis, Missouri, USA CA‐630, 1 mmol/L dithiothreitol [DTT], 100 μg/mL phenylmethylsulfonyl fluoride [PMSF], and a protease Inhibitor cocktail [Amresco]), and the beads were incubated with lysate supernatants for 4 h at 4°C in dilution buffer (50 mmol/L Tris‐HCl pH 8, 150 mmol/L NaCl, 0.1 mmol/L ethylenediaminetetraacetic acid [EDTA], 1 mmol/L DTT, 100 μg/mL PMSF, and a protease Inhibitor cocktail [Amresco]), and washed three times with wash buffer (50 mmol/L Tris‐Hcl pH 8, 150 mmol/L NaCl, 0.1 mmol/L EDTA). Aliquots of input, nonbound, washes, and bound samples were analyzed by immunoblotting.
Protease inhibitor cocktail
Manufactured by Avantor
Sourced in United States, China
Protease inhibitor cocktail is a formulation designed to inhibit the activity of proteases, which are enzymes that break down proteins. This product can be used to preserve the integrity of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (6 mentions)
Dc protein assay, by Bio-Rad (4 mentions)
Ripa buffer, by Applygen (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Gel doc xr system, by Bio-Rad (3 mentions)
Las 3000, by Fujifilm (3 mentions)
Phosphatase inhibitor cocktail, by Merck Group (3 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (3 mentions)
Odyssey clx imaging system, by LI COR (3 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Avantor (3 mentions)
Dithiothreitol (dtt), by Avantor (2 mentions)
Protein marker, by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Glycine, by Merck Group (2 mentions)
Imidazole, by Merck Group (2 mentions)
Scion image software, by Techcomp Instruments (2 mentions)
Ecl reaction, by Cytiva (2 mentions)
Anti cb1r, by Cayman Chemical (2 mentions)
90 protocols using protease inhibitor cocktail
1
CHO Cell Transfection and GFP-Trap Assay
2
Purification and Interaction of UHRF1 and LSH
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To express UHRF1 in bacteria, PCR products encoding UHRF1 were ligated into pGEX4T-1 vector. GST-UHRF1 fusion protein was expressed and purified from E. coli. To purify FLAG-tagged LSH protein from mammalian cells, the 293T cells were transfected with plasmid encoding FLAG-LSH for 48 h. The cells were collected and lysed in high salt Lysis buffer (25 mM Tris–HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1× protease inhibitor cocktail, 1 mM DTT). FLAG-LSH protein was then captured with anti-FLAG M2-affinity beads and eluted with FLAG-peptide elution buffer (100 μg/ml FLAG-peptides, 50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM EDTA, 1× protease inhibitor cocktail, 1 mM DTT). For pulldown assay to test in vitro binding of UHRF1 and LSH, 1 μg of purified FLAG-LSH and 2μg of bead-bound GST-UHRF1 beads were incubated in pulldown binding buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0. 1% Triton X-100, 1 mM EDTA, 8% glycerol, 1× protease inhibitor cocktail (MCE), and 1 mM DTT (Amresco)) for 6 h at 4°C. The resulting beads were washed three times with ice-cold pulldown wash buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0. 1% Triton X-100, 1 mM EDTA, 1× protease inhibitor cocktail (MCE) and 1 mM DTT (Amresco)). After extensive washing, complexes were boiled in 1 × SDS loading buffer and analyzed by western blotting and Coomassie blue staining.
3
Protein Extraction from M. smegmatis
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M. smegmatis cells were pelleted; rinsed three times with Middlebrook 7H9, 0.2% glycerol, and 0.05% Tween 80 at 4°C; resuspended in PBS + 2% SDS + protease inhibitor cocktail (VWR; catalog number 97063-972); and transferred to 2-ml disruption tubes (OPS Diagnostics; 100-μm zirconium lysing matrix, molecular grade). Cultures were lysed using a FastPrep-24 5G instrument (MP Biomedical) using four cycles of 6.5 m/s for 30 s with 1 min on ice between cycles. Samples were clarified by centrifugation at 21,130 × g at 4°C for 10 min, and the supernatant containing protein was recovered and stored at –20°C. Protein concentrations were calculated using the Pierce BCA protein assay (Thermo Scientific; catalog number 23225) according to the manufacturer’s instructions.
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4
Serum Metabolic Profiling in Mice
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Blood was collected from the mice via cardiac puncture, serum removed and stored at −80°C. Protease inhibitor was added to sera (Protease Inhibitor Cocktail, VWR Life Science Amresco) and analyzed for metabolic hormones (Mouse Metabolic Array) and a panel of 31 chemokines/cytokines (Mouse Cytokine Array/Chemokine Array 31-Plex) by addressable laser bead immunoassay by Eve Technologies (evetechnologies.com; Calgary, AB, Canada).
5
Evaluation of Adenosine A2A Receptor
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Materials were ordered from Sigma-Aldrich (St. Louis, MO, USA), except for RAW 264.7 and HEK-293 cell lines (ATCC, New York, NY, USA), HEK-293 transgenic cell line expressing Flag-A2AR (provided by Francesco Ciruela, Department of Pathology and Experimental Therapeutics, University of Barcelona, Spain), DMEM (LM-D1111, GENTAUR Europe BVBA, Kampenhout, Belgium), FBS (FB-1090, GENTAUR Europe BVBA, Kampenhout, Belgium), T75 cell culture dish (Z707546, TPP, Trasadingen, Switzerland), Falcon Multiwell tissue culture plates (351146, BD Biosciences, San Jose, CA, USA), TRI reagent (TR118, Molecular Research Center, Cincinnati, OH, USA), Maxima SYBR Green/ROX qPCR Master Mix (2X) (K0222, Thermo Scientific, Waltham, MN, USA), SDS-PAGE (4568034, Bio-RAD, Hercules, CA, USA), West Pico Super Signal ECL Western Blotting Detection Reagent (34580, Thermo Fisher, Waltham, MA, USA), 8 wells tissue culture treated glass chamber slides (354118, Falcon, Chicago, IL, USA), 96-well Cell Carrier Ultra plate (6055302, Perkin Elmer, Waltham, MA, USA), CGS21680 (1063, Tocris, Minneapolis, MN, USA), phenylmethylsulfonyl fluoride (PMSF-RO, Merck, Darmstadt, Germany), protease inhibitor cocktail (M221, VWR International, Radnor, PA, USA).
6
Homogenization and Protein Extraction from Human Cortical Tissue
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0.3 g slow frozen human cortical ribbon from each case was homogenized for 30 passes in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris HCl, pH 7.4; NP-40, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1mM PMSF, and 1X Protease Inhibitor Cocktail, Mammalian [VWR, Radnor, PA] added at time of use.). Alternatively, 600 ug of mitochondrial fraction pellets from each case was resuspended in 300 ul RIPA buffer. All RIPA suspensions were sonicated for 4 min in a water bath sonicator, held on ice for 30 min with vortexing every 5 min, and centrifuged at 15,000 X g for 10 min. Supernatants were saved, protein concentrations determined using a Pierce Bradford assay, and 40 ug aliquots stored at -80°C.
7
Protein Extraction and Immunoblotting of B-ALL Cells
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B-ALL cells were harvested and lysed in M-PER buffer (Invitrogen) containing 1% protease inhibitor cocktail (VWR). Protein concentration was determined by Bradford protein assay. Proteins were separated by 4-12% Bis-Tris protein gels (Invitrogen) and transferred to PVDF membranes (Invitrogen). The antibodies (Abs) used are listed in Table S2
8
Extraction and Characterization of A. satureioides
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A. satureioides was acquired from Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas (CPQBA) da Universidade Estadual de Campinas (São Paulo, Brazil), via a sample deposited in the herbarium with the number 308. Egg yolk lecithin and medium-chain triglycerides were purchased from Lipoid (Ludwigshafen, Germany), polysorbate 80 was acquired from Vetec (Rio de Janeiro, Brazil), and vitamin E was procured from Alpha Química (Cachoeirinha, Brazil). Ultra-fast liquid chromatography (UFLC) required the following reagents: methanol (J.T. Baker, Philipsburg, NJ, USA), acetonitrile (Tedia, Rio de Janeiro, Brazil), and phosphoric acid (Merck, Rio de Janeiro, Brazil). Carbopol® Ultrez 20 was kindly donated by Lubrizol do Brasil Aditivos Ltd.a (São Paulo, Brazil). Albumin for enzyme-linked immunosorbent assay (ELISA) was purchased from Calbiochem (San Diego, CA, USA). Normal goat serum was obtained from R&D Systems (Minneapolis, MN, USA). Protease inhibitor cocktail was purchased from VWR Life Science (Radnor Township, PA, USA). Xylazine and ketamine were purchased from Ceva (Paulínea, Brazil). Ketoprofen was obtained from Merial (Paulínea, Brazil). Finally, thiopental was obtained from Cristália (Itapira, Brazil).
9
Fluorometric Assay for Glucosidase Activity
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Enzyme assay was performed as previously described (21–24). Briefly, extracted tissues
were harvested, rinsed in PBS, and flash frozen in liquid nitrogen. Whole organs were
homogenized in molecular grade water with protease inhibitor cocktail (VWR), using a
FastPrep24 (MP Biomedicals) followed by three freeze/thaws. Non-homogenized materials were
pelleted, and clarified lysate was maintained at −80 °C. Five µl of each sample was
incubated with 4-methylumbelliferyl-α-D-glucosidase (Millipore-Sigma) for 1 h at pH 4.3.
Fluorescence resulting from substrate cleavage was detected with a Tecan Multimode Plate
Reader and coordinating Magellan Software. Sample readings were compared to a standard
curve generated from 4-methylumbelliferone (Millipore-Sigma). All samples were normalized
to protein levels determined by DC Protein Assay (Bio-Rad), performed in accordance with
the manufacturer’s protocol.
were harvested, rinsed in PBS, and flash frozen in liquid nitrogen. Whole organs were
homogenized in molecular grade water with protease inhibitor cocktail (VWR), using a
FastPrep24 (MP Biomedicals) followed by three freeze/thaws. Non-homogenized materials were
pelleted, and clarified lysate was maintained at −80 °C. Five µl of each sample was
incubated with 4-methylumbelliferyl-α-D-glucosidase (Millipore-Sigma) for 1 h at pH 4.3.
Fluorescence resulting from substrate cleavage was detected with a Tecan Multimode Plate
Reader and coordinating Magellan Software. Sample readings were compared to a standard
curve generated from 4-methylumbelliferone (Millipore-Sigma). All samples were normalized
to protein levels determined by DC Protein Assay (Bio-Rad), performed in accordance with
the manufacturer’s protocol.
10
Nuclear Extraction and Purity Verification
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Nuclear extraction was performed by incubation of cells for 20 min on ice with nuclei isolation buffer (10 mM Tris-HCl at pH 7.8, 4 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 1% Triton X-100, 0.25 M Sucrose, 10 mM NaF, 5 mM Sodium Pyrophosphate, 2 mM Sodium Orthovanadate); after centrifugation to isolate nuclei, they were washed with isolation buffer without Triton X-100 and centrifuged two times. Each buffer was supplemented with protease inhibitor cocktail (AMRESCO).
Total cell extracts, cytosolic and nuclear fractions were analyzed by western blot for purity determination. Antibodies against LDH-A and Lamin B1 were used as purity control of cytosolic and nuclear fraction, respectively.
Total cell extracts, cytosolic and nuclear fractions were analyzed by western blot for purity determination. Antibodies against LDH-A and Lamin B1 were used as purity control of cytosolic and nuclear fraction, respectively.
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