HEK 293T cells were grown in 6 cm dishes and treated with 15 μM lovastatin (M2147-25MG, Sigma) for about 2 h. Fresh medium containing 400 μCi of mevalonolactone RS-[5-3H] (ART 0315A, ARC) and 15 μM lovastatin was added. Cells were then transiently transfected with FLAG-tagged FBXL2(C417S/C419S) and FBXL2(C420S). After overnight incubation, cells were harvested and lysed with Nonidet P-40 substitute (Roche)-based lysis buffer containing protease inhibitor cocktail (Roche) and sonicated (Sonic Dismembrator, Fisher Scientific). A portion of the lysate was taken as input and the rest was clarified using Protein G Agarose (Invitrogen) followed by overnight immunoprecipitation with anti-flag M2 affinity gel (A2220-Sigma). After washing the M2 gel, elution was carried out with 2X Bolt LDS sample buffer (novex). The samples were run on Bolt 4–12% Bis-Tris Plus gel (Invitrogen) and transferred onto PVDF membrane (Millipore). The membrane was placed under a phosphor screen (28-9564-82, Cytiva) for two weeks and the screen was exposed in a phosphor imager (Amersham Typhoon, GE) for [3H] detection. The PVDF membrane was finally immunoblotted for FLAG-tagged FBXL2(C417S/C419S) and FBXL2 (C420S) expression using rabbit anti-FLAG antibody (F7425-Sigma).
Nonidet p 40 substitute
Manufactured by Roche
Sourced in Germany
Nonidet P-40 substitute is a non-ionic detergent used in various laboratory applications, such as cell lysis, protein extraction, and immunoassays. It is a milder, less denaturing alternative to Triton X-100. The product effectively solubilizes proteins while maintaining their native structure and functionality.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Protein g agarose, by Thermo Fisher Scientific (2 mentions)
Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Bolt lds sample buffer, by Thermo Fisher Scientific (2 mentions)
F7425, by Merck Group (2 mentions)
Sonic dismembrator, by Thermo Fisher Scientific (2 mentions)
M2147 25mg, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Amersham typhoon, by GE Healthcare (2 mentions)
Mammalian protease inhibitor cocktail, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Resazurin solution, by Merck Group (1 mentions)
Dm4000b, by Leica (1 mentions)
X well pca tissue culture chambers, by Sarstedt (1 mentions)
Tris hcl ph7, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
10 protocols using nonidet p 40 substitute
1
Tracking Mevalonate Metabolism in FBXL2 Mutants
2
Pervanadate Disruption of Kinase-Phosphatase Balance
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Basal kinase/phosphatase balance was disrupted by inhibition of phosphatase activity with a serial dilution of pervanadate. Pervanadate was generated immediately before the assay by incubating 10 mM sodium orthovanadate with 0.7% H2O2 at room temperature for 10 min. H2O2 was inactivated by the addition of 20 μg/mL catalase at room temperature for 10 min. A 24-step, 1.25-fold serial dilution of pervanadate was made up in serum-free medium starting at 1 mM. Harvested transfectants were plated at 300,000 cells per well in 96-well assay plates in a volume of 60 μL/well and treated with 60 μL/well pervanadate serial dilutions for 30 min at 37°C in a tissue culture incubator. Reactions were terminated and cells were lysed by adding 120 μL/well lysis buffer containing 2% Nonidet P-40 substitute (Roche, Indianapolis, IN), Mammalian Protease Inhibitor Cocktail (Sigma-Aldrich), and 250 μM sodium orthovanadate in TBS (20 mM Tris, pH 7.5, 150 mM NaCl). Cellular debris was removed by centrifugation and lysates were transferred to wells of previously prepared immunosorbent capture plates.
3
Tracking Mevalonate Metabolism in FBXL2 Mutants
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HEK 293T cells were grown in 6 cm dishes and treated with 15 μM lovastatin (M2147-25MG, Sigma) for about 2 h. Fresh medium containing 400 μCi of mevalonolactone RS-[5-3H] (ART 0315A, ARC) and 15 μM lovastatin was added. Cells were then transiently transfected with FLAG-tagged FBXL2(C417S/C419S) and FBXL2(C420S). After overnight incubation, cells were harvested and lysed with Nonidet P-40 substitute (Roche)-based lysis buffer containing protease inhibitor cocktail (Roche) and sonicated (Sonic Dismembrator, Fisher Scientific). A portion of the lysate was taken as input and the rest was clarified using Protein G Agarose (Invitrogen) followed by overnight immunoprecipitation with anti-flag M2 affinity gel (A2220-Sigma). After washing the M2 gel, elution was carried out with 2X Bolt LDS sample buffer (novex). The samples were run on Bolt 4–12% Bis-Tris Plus gel (Invitrogen) and transferred onto PVDF membrane (Millipore). The membrane was placed under a phosphor screen (28-9564-82, Cytiva) for two weeks and the screen was exposed in a phosphor imager (Amersham Typhoon, GE) for [3H] detection. The PVDF membrane was finally immunoblotted for FLAG-tagged FBXL2(C417S/C419S) and FBXL2 (C420S) expression using rabbit anti-FLAG antibody (F7425-Sigma).
4
Nucleosome Binding Experiments with Xenopus Histones
5
Chagas Disease Chemotherapy Protocol
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To set the new assay up, the drugs currently marketed for the chemotherapy of Chagas disease were selected as reference compounds: BZ and NFX. Pure drugs were provided by Roche, Brazil, Bayer and Germany, respectively, and they were dissolved in dimethyl sulfoxide (DMSO; Stanton®, Buenos Aires, Argentina) in stock solutions at 20 mg/ml. DMSO concentration never exceeded 0.2% in plate wells, with no effect on the host cell viability nor parasite replicative growth [16 (link)]. Roswell Park Memorial Institute (RPMI)-1640 with or without phenol red (Sigma; R6504 or R8755, respectively). Sodium bicarbonate (Emeve®; P1551-500). Gentamicin (Sigma; G1397). Penicillin–streptomycin (Sigma; P4333). Resazurin solution (Sigma, R7017). CPRG (Roche Diagnostics GmbH; 10 884 308 001). Nonidet™ P-40 substitute (Roche Diagnostics GmbH; 11 754 599 001). Heat-inactivated, UV-irradiated foetal bovine serum (FBS) (Natocor® or Internegocios S.A, Argentina). Geneticin (G418; Life Technologies).
6
Immunofluorescence Microscopy of Aflibercept and Bevacizumab
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Confluent iBREC were cultivated on fibronectin-coated two-chamber slides (x-well PCA Tissue Culture Chambers; Sarstedt, Nuembrecht, Germany) and exposed to aflibercept or bevacizumab for 4 h as described above [7 (link), 12 (link)]. After methanol-fixation (10 min at − 20 °C), cells were permeabilized for 10 min in 0.25% Nonidet P-40 substitute (Roche, Mannheim, Germany) diluted in PBS without Ca2+/Mg2+ ions (PBSd). After incubation of the slides in blocking solution (10% ImmunoBlock, Roth, Karlsruhe, Germany) for 60 min at room temperature, they were treated for 30 min with AlexaFluor594-conjugated antibodies binding to human IgG to detect aflibercept or bevacizumab. Slides were then exposed to primary antibodies recognizing endogenous proteins of interest for 1 h at room temperature, and subsequently to appropriate AlexaFluor488-conjugated goat F(ab’)2 fragments for 30 min. Primary and secondary antibodies were always diluted in 1% ImmunoBlock/PBSd. For examination by fluorescence microscopy (DM4000B, FW4000, Leica, Wetzlar, Germany), cells were embedded in ProLong Gold/Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) [7 (link), 12 (link), 27 (link)].
7
STING Activation and Protein Purification
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293T cells were transduced with pTRIP-hPGK-Blast-2A-STING-HA and pTRIP-hPGK-Hygro-2A-FLAG-Ubiquitin and selected with 15 µl Blasticidin (Invivogen) and 320 µg/ml Hygromycin (Invivogen) for one week. 1 million cells/well were plated in a six-well plate the day prior to the stimulation. Cells were then stimulated with 300 µl cGAMP permeabilization buffer containing 1 µg/ml cGAMP for 10 min, washed with 3 ml of warm medium, and the medium was replaced. 3 wells per condition were harvested 2 h post stimulation, washed with PBS and lysed with 550 µl of RIPA buffer for 10 min on ice. Lysates were cleared at 16,000 × g for 10 min at 4 °C. 10% of the lysate was saved as input. The remaining lysates were incubated with 150 µl Pierce Anti-DYKDDDDK Magnetic beads (Thermo Fisher) O/N at 4 °C with constant rotation. Beads were washed three times with a buffer containing 10 mM Tris-Hcl pH7.5 (Thermo Fisher), 2 mM EDTA (Thermo Fisher), 1% Nonidet-P40 Substitute (Roche) and 50 mM NaCl (Thermo Fisher), and two times with RIPA buffer. Proteins were eluted by adding 150 µl of non-reducing Laemmli (Boston bioproducts) containing 20 mM DTT (Thermo Fisher) and boiled for 20 min. Input was diluted with 2X sample buffer (Sigma).
8
Liver Triglyceride Quantification Protocol
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Triglyceride Quantification Kit (Sigma, St-Louis, MO, USA) was used to determine liver TG by colorimetric method. Liver tissue samples were homogenized in 5% Nonidet P 40 Substitute (Roche, Mannheim, Germany). The samples were heated to 100℃ in water bath for 5 minutes or until the Nonidet P 40 became cloudy, and then cooled to room temperature. Heating was reported once more to solubilize all TG. The samples were centrifuged for 2 minutes at maximum speed to remove insoluble material, and then diluted 100-fold with deionized water before assay.
9
Monitoring Nucleosome Binding Kinetics
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Histone H4(A15C) was labeled with Cy3 maleimide and reconstituted into nucleosomes (26 (link)). Changes in Cy3 fluorescence intensity were monitored in a Horiba Jobin Yvon Fluorolog fluorometer with the sample chamber set at 25°C, excitation monochromator at 510 nm (5 nm slit width), and emission monochromator at 565 nm (5 nm slit width). Increasing amounts of Chd1 were titrated into solutions containing 5 nM nucleosome in 20 mM HEPES, pH 7.5; 5 mM MgCl2; 0.1 mM EDTA; 5% (w/v) sucrose; 1 mM DTT; 0.02% Nonidet P40 Substitute (Roche); 0.1 mg/ml BSA; 1 mM adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) and 100 mM KCl. Titrations with LacI contained 500 nM LacI. The binding data was fit in KaleidaGraph (Synergy) to the following binding isotherm using nonlinear least squares regression,
where Y is the signal intensity, X is the concentration of Chd1, AX are the signal amplitudes, KX are the dissociation constants, N is the nucleosome concentration and C is the signal from nucleosome alone. Titrations for each experimental condition were performed three or more times, with the average 1/K1/2 values and standard deviations reported in Figure3 .
where Y is the signal intensity, X is the concentration of Chd1, AX are the signal amplitudes, KX are the dissociation constants, N is the nucleosome concentration and C is the signal from nucleosome alone. Titrations for each experimental condition were performed three or more times, with the average 1/K1/2 values and standard deviations reported in Figure
10
Single-Cell ATAC-Seq for CD8+ T Cells
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Nuclei isolation was performed using the 10x Nuclei Isolation for Single Cell ATAC Sequencing protocol for low cell input. In brief, dextramer sorted CD8 + T cells were pooled for each genotype from individual mice with equal cell numbers. Cells were resuspended in chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.1% Tween 20, 0.1% Nonidet P40 Substitute (Roche), 0.01% Digitonin (Sigma), 1% BSA (Miltenyi) in nuclease free water) and incubated on ice for 3 min. Cells were washed, resuspended in Nuclei Buffer (10x) and 6.000-10.000 nuclei were used for subsequent single cell ATAC library preparation according to the 10x genomics ChromiumNext GEM Single Cell ATAC v1.1 user guide.
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