Cells were lysed in a nucleus extraction buffer (NB; 0.25 M sucrose [Sigma-Aldrich, S0389], 10 mM Tris [Sigma-Aldrich, 93352], pH 8, 10 mM MgCl2 [Sigma-Aldrich, 442,615], 1 mM EDTA [Sigma-Aldrich, 03609], 1% Triton X-100 [Sigma-Aldrich, T9284], 0.5 mM DTT [Bio-Rad Laboratories, 1,610,611) containing 10 mM NaF (Sigma-Aldrich, S7920), 1 mM Na3VO4 (Sigma-Aldrich, 567540) and protease inhibitor cocktail (Roche, P8849). After centrifugation at 600 x g for 10 min at 4°C, supernatants were transferred to new tubes and pellets containing nuclei were resuspended with NB and centrifuged at 600 x g for 10 min at 4°C. Pellets were then lysed in RIPA buffer (50 mM Tris [Sigma-Aldrich, 93,352], pH 8.0, 150 mM NaCl [Sigma-Aldrich, S9625], 1% NP40 [Sigma-Aldrich, 74,385], 0.1% SDS [Sigma-Aldrich, 8170341000], 0.5% sodium deoxycholate [Sigma-Aldrich, D6750]) supplemented with 10 mM NaF (Sigma-Aldrich, S7920), 1 mM Na3VO4 (Sigma-Aldrich, 567540) and protease inhibitor cocktail (Roche, P8849) and processed for SDS-PAGE.
Na3vo4
Manufactured by Merck Group
Sourced in United States, Germany, China, Switzerland
Na3VO4 is a chemical compound that is commonly used as a laboratory reagent. It is a white, crystalline solid that is soluble in water. Na3VO4 serves as a source of the vanadate ion (VO4^3-), which is useful in various chemical processes and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (34 mentions)
Leupeptin, by Merck Group (29 mentions)
Aprotinin, by Merck Group (26 mentions)
Protease inhibitor cocktail, by Merck Group (24 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (20 mentions)
Dithiothreitol (dtt), by Merck Group (18 mentions)
Triton x 100, by Merck Group (16 mentions)
Protease inhibitor cocktail, by Roche (15 mentions)
Pvdf membrane, by Merck Group (13 mentions)
Np 40, by Merck Group (12 mentions)
Nonidet p 40, by Merck Group (12 mentions)
Sodium chloride (nacl), by Merck Group (11 mentions)
Tris hcl, by Merck Group (10 mentions)
β glycerophosphate, by Merck Group (10 mentions)
Hepes, by Merck Group (9 mentions)
Pepstatin, by Merck Group (9 mentions)
Complete protease inhibitor cocktail, by Roche (9 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (9 mentions)
Ethanol, by Merck Group (8 mentions)
β actin, by Merck Group (7 mentions)
196 protocols using na3vo4
1
Nuclear Extraction and Protein Isolation
2
Western Blot Analysis of Protein Lysates
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Cells were washed with cold PBS containing 1 mM Na3VO4 (Sigma) and lysed in a solution containing 1% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8), 1.5 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 10 mM Na4P2O7, and complete protease inhibitors (Roche) (56) . After incubation at -70°C, lysates were centrifuged for 15 min to remove insoluble material. After protein quantiZication, 30 μg of sample was boiled (100ºC) for 6 min in SDS sample buffer and migrated in SDS-PAGE gels. Proteins were transferred onto Hybond-ECL membranes (Amersham Biosciences, 10600003), blocked for 2 h in TBS1X-0.1% Tween containing 5% BSA, incubated with primary antibodies (diluted in TBS1X-0.1% Tween containing 5% BSA) overnight at 4°C, and thence with suitable HRP-conjugated secondary antibodies (GE Healthcare NA931V and NA934V) for 1 h at room temperature. Washed membranes were incubated in ECL (GE Healthcare, RPN2106) and immunodetection was achieved via chemiluminescence (62) . Densitometric analysis of Western blot bands was done using the AlphaEase FC software (Alpha Innotech) with β-actin used as loading control.
3
Optimized Protein Extraction from HepG2 Cells
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Stable cell lines were grown to 80% confluence in 150 mm Petri dishes. Sample preparation and protein extraction were optimized using non-transfected HepG2 cells. The optimizations regarded cell harvesting (scraping vs. in situ lysis), cell disruption (freeze/thaw vs. tip sonication), and washing and lysis buffer. All chemicals were purchased from GE Healthcare (Munich, Germany) unless otherwise specified. Sample buffer components tested included the reducing/oxidizing agent (DTT vs. HED), spermine (Sigma-Aldrich, Taufkirchen, Germany), benzonase (Sigma-Aldrich), as well as the detergent assortment of the ProteoPrep detergent sampler kit (Sigma-Aldrich). Following the optimized protocols, cells were washed three times with Tris buffered sucrose solution containing protease and phosphatase inhibitors (2 mM PMSF—Sigma-Aldrich, 1 mM NaF—Sigma-Aldrich and 0.2 mM Na3VO4—Merck, Darmstadt, Germany) and detached with a cell-scraper in the same buffer. Cells were pelleted, then resuspended in 1 ml lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 25 mM spermine and 0.5% Pharmalyte 3–10) and disrupted by tip sonication on ice (5 x 30 second cycles 90% of time at 70% power). After 1 h incubation on ice, the protein extract was centrifuged for 30 min at 40000 x g and 4°C and proteins were quantified using RC-DC Quant kit (Bio-Rad, Munich, Germany).
4
Biochemical Analysis of Sphingolipid Metabolism
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JTE-013, C6 NBD dihydroceramide (NBD-C6-dhCer) and (2S,3R)-2-ammonio-3-hydroxy-5-((2-oxo-2H-chromen-7-yl)oxy)pentyl hydrogen phosphate were from Cayman Chemical (Ann Arbor, MI). SKi (4-[[4-(4-chlorophenyl)-2-thiazolyl]amino]phenol)40 (link), dithiothreitol (DTT), fatty acid-free bovine serum albumin (BSA), human cytochrome B5 (CYB5, Sigma #C1427, E.C.1.6.2.2), FLAG-agarose beads, FLAG peptide, dodecyl maltoside (DDM), SYPRO-RUBY, Complete© EDTA-free protease inhibitors (CPI), Na3VO4, pyridoxal 5′ phosphate, trifluoracetic acid and acetonitrile were from Merck (Frenchs Forest, NSW, Australia). MG132 was purchased from Selleck Chem (Houston, TX). HPLC columns and vials were purchased from Phenomenex (Torrence, CA, USA).
5
Phosphorylation Measurement via ELISA
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Lysates for enzyme-linked immunosorbent assay (ELISA) methods were obtained using a lysis buffer pH 7.6 with EDTA-free protease inhibitors (Roche Diagnostics, Mannheim, Germany) and Na3VO4 (Merck, Darmstadt, Germany), as phosphatase inhibitor, and a Retsch ball mills homogenizer (Retsch GmbH, Haan, Germany) at a vibrational frequency of 30 Hz during 1 min. The phosphorylation of this receptor was measured by using an ELISA kit from Assay Solution (Woburn, MA, USA). Hepatic lysates were incubated for 2 h at room temperature (RT) and after washing, a detection antibody coupled to biotin was incubated in the microplate during 2 h at the same temperature. A complex streptavidin-horseradish peroxidase (HRP) was added and finally, after washing, tetramethylbenzidine was incubated until the lecture of the absorbance at 450 nm. The intra- and inter-assay coefficients of variation were lower than 10%.
6
Diverse Chemical Inhibitors in Microbial Growth
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With the exception of hygromycin B (Panreac Applichem), all chemicals were from Sigma-Aldrich: paromomycin, streptomycin, Na2CrO4, Na2MoO4, Na3VO4, 4,4′-diisothio-cyanatostilbene-2,2′-disulphonate (DIDS), sodium malonate, sodium oxalate, NaHCO3, Na2SeO4, Na2S2O3, Na2WO4, Na2S4O6. With the exception of DIDS (in 0.1 M potassium bicarbonate), stock solutions of all chemicals used to inhibit growth were prepared in distilled water and added to growth media at the specified final concentrations. All stock solutions were filter-sterilized before additions to media.
7
Synthesis of Luminescent Nanoparticles
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Gadolinium(III) nitrate hexahydrate (Gd(NO3)3·6H2O, Aldrich, 99.9%), europium(III)
nitrate pentahydrate (Eu(NO3)3·5H2O, Aldrich, 99.9%), sodium orthovanadate (Na3VO4, Aldrich, 99.9%), poly(acrylic acid) (PAA, Aldrich, average Mw ≈ 1800), ethylene glycol (EG), zirconium(IV) n-propoxide 70% in 1-propanol (Aldrich), tetraethyl orthosilicate
(TEOS, Aldrich), triblock copolymers Pluronic F127 (Mw ≈ 12 600), 2,4-pentanedione (acetylacetonate,
acac, AlfaAesar), SiO2 (LUDOX TMA, Aldrich), absolute ethanol,
methanol, HCl (3.571 mol/L, Panreac), and Milli-Q water.
nitrate pentahydrate (Eu(NO3)3·5H2O, Aldrich, 99.9%), sodium orthovanadate (Na3VO4, Aldrich, 99.9%), poly(acrylic acid) (PAA, Aldrich, average Mw ≈ 1800), ethylene glycol (EG), zirconium(IV) n-propoxide 70% in 1-propanol (Aldrich), tetraethyl orthosilicate
(TEOS, Aldrich), triblock copolymers Pluronic F127 (Mw ≈ 12 600), 2,4-pentanedione (acetylacetonate,
acac, AlfaAesar), SiO2 (LUDOX TMA, Aldrich), absolute ethanol,
methanol, HCl (3.571 mol/L, Panreac), and Milli-Q water.
8
Protein Extraction and Western Blotting
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Cells were homogenized in RIPA buffer supplemented with protease inhibitors (Complete tablets, Roche Diagnostics, Cat# 11836153001), 1 mM AEBSF (ThermoFisher Scientific, Cat# 50-213-115), and 1 mM Na3VO4 (Sigma, Cat# S6508-10G). Antibodies for western analyses included: phospho-RON (R&D Systems, Cat# AF1947), RON-β (C-20) (Santa Cruz Biotechnology, Cat# SC-322), β-CATENIN (Cell Signaling Technology, Cat# 9582S and BD Biosciences, Cat# 610154), FLAG (Sigma, Cat# F1804), HGFL (T-19) (Santa Cruz Biotechnology, Cat# SC-6090), phospho-NF-κB p65 (Cell Signaling Technology, Cat# 3033S), NF-κB p65 (Cell Signaling Technology, Cat# 8242S), TUBULIN (Santa Cruz Biotechnology, Cat# SC-5286), and C4-ACTIN (Cincinnati Children's Hospital Medical Center). Peroxidase-conjugated secondary antibodies were applied and membranes were developed using Pierce ECL2 Western Blotting substrate (ThermoFisher Scientific, Cat# PI80196×3).
9
Protein Analysis of Osteoclasts and Macrophages
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For protein content analysis, osteoclast, or macrophage cultures were washed and then lysed in a Triton-based lysis buffer containing 100 mM NaCl, 30 mM Na-HEPES (pH 7.4), 20 mM NaF, 1 mM Na-EGTA, 1% Triton X-100, 1 mM benzamidine, freshly supplemented with 0.1 U/ml Aprotinin, 1:100 Mammalian Protease Inhibitor Cocktail, 1:100 Phosphatase Inhibitor Cocktail 2, 1 mM PMSF, and 1 mM Na3VO4 (all from Sigma-Aldrich). Insoluble material was removed, the lysate supernatants were supplemented with 4× Laemmli's sample buffer and boiled for 10 min. Whole cell lysates were run on SDS-PAGE, electroblotted to nitrocellulose membranes, and then processed for immunoblotting with antibodies against Syk (N19; Santa Cruz) or β-actin (Clone AC-74; Sigma-Aldrich). After incubation with peroxidase-labeled secondary antibodies (GE Healthcare), the signal was developed using the ECL system (GE Healthcare) and exposed to X-ray film. X-ray films were then scanned and processed with Adobe Photoshop.
10
Protein Extraction and Fractionation Protocol
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HeLa, HEK293T and ETNA total extracts were obtained as follows. After ×3 washing steps in ice cold phosphate-buffered saline (PBS) (Lonza, Basel, Switzerland), cells were lysed in homemade RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2.5 mM Na3VO4, 5 mM NaF, 1 mM DTT, all reagents purchased from Sigma-Aldrich) complemented with protease inhibitor cocktail (Sigma-Aldrich). Samples were collected and centrifuged at 13,000g for 10 min at 4 °C; supernatants were saved.
For cytosol/mitochondria fractionation, cells were lysed by mechanical breaking (~100 potter strokes) into a detergent-free buffer (0.25 M sucrose, 10 mM HEPES pH 7.5, 1 mM EDTA). A first centrifugation (600g for 10 min at 4 °C) was employed to pellet nuclei; subsequently, supernatants were recentrifuged at 12,000g for 15 min at 4 °C to separate a cytosolic upper phase from a mitochondrial pellet. The latter was eventually lysed in RIPA buffer.
All protein extracts were quantified through the Bradford Protein Assay (Bradford solution from Bio-Rad Laboratories) according to the manufacturer’s protocols.
For cytosol/mitochondria fractionation, cells were lysed by mechanical breaking (~100 potter strokes) into a detergent-free buffer (0.25 M sucrose, 10 mM HEPES pH 7.5, 1 mM EDTA). A first centrifugation (600g for 10 min at 4 °C) was employed to pellet nuclei; subsequently, supernatants were recentrifuged at 12,000g for 15 min at 4 °C to separate a cytosolic upper phase from a mitochondrial pellet. The latter was eventually lysed in RIPA buffer.
All protein extracts were quantified through the Bradford Protein Assay (Bradford solution from Bio-Rad Laboratories) according to the manufacturer’s protocols.
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