Nupage novex gel

Manufactured by Thermo Fisher Scientific

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The NuPAGE Novex gel is a pre-cast polyacrylamide gel designed for electrophoretic separation of proteins. It is used for high-resolution analysis and purification of proteins.

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NuPAGE (743 citations) NuPAGE gels (529 citations) NuPAGE Novex (59 citations) Novex gels (17 citations) SDS NuPAGE Novex 4–12% gels (9 citations) NuPAGE Novex 4–12% Bis-Tris ZOOM Gels (3 citations) NuPAGE® Novex® Bis-Tris 15 well precast gels (3 citations) Novex NuPAGE 10% Bis-Tris Protein gels (2 citations) NuPAGE Novex 4–12% or 12% Bis-Tris mini gels (2 citations) NuPAGE® Novex 4–12% (w/v) Bis-Tris SDS-PAGE gels (2 citations)

37 protocols using nupage novex gel

Immunoblotting of Phosphorylated RET

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Cells were lysed in ice-cold RIPA buffer (20 mM Tris pH 7.4, 137 mM NaCl, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA pH 8.0) containing protease and phosphatase inhibitors.
Proteins were quantified using a modified Bradford assay (Bio-Rad). Protein samples were boiled in NuPAGE LDS sample buffer (Invitrogen), separated by NuPAGE Novex Gels with the appropriate running buffer (Invitrogen), then transferred onto nitrocellulose filters, and immunoblotted with the indicated antibodies. Anti-RET (C-20), anti-RET (H300) and anti-phospho-RET are from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-MAP kinase (ERK1/2), anti-MAP kinase activated (pERK1/2), anti-vinculin and anti-β-tubulin are from Sigma Aldrich (Saint Louis, Missouri, USA).
Densitometric analyses were performed by the Quantity One 4.6.6 software (Bio-Rad, Hercules, CA).

Colombo C., Minna E., Rizzetti M.G., Romeo P., Lecis D., Persani L., Mondellini P., Pierotti M.A., Greco A., Fugazzola L, & Borrello M.G. (2015). The modifier role of RET-G691S polymorphism in hereditary medullary thyroid carcinoma: functional characterization and expression/penetrance studies. Orphanet Journal of Rare Diseases, 10, 25.

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Western Blot Analysis of Proteins

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Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis analysis was carried out using NuPAGE Novex gels (Invitrogen), and the proteins were transferred onto PVDF membranes (Invitrogen), followed by blocking with 5% skim milk for 1 h, and an incubation with primary antibodies overnight at 4°C. The membranes were washed with TBST (25 mM Tris, pH 7.4, 150 mM NaCl, 2 mM KCl and 0.01% Tween 20), and incubated with anti-rabbit or mouse IgG horseradish peroxidase-conjugated antibodies (GE Healthcare) for 1 h at room temperature, and then washed with TBST buffer. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology, Rockford, IL, USA).

Han S.J., Lee B.C., Yim S.H., Gladyshev V.N, & Lee S.R. (2014). Characterization of Mammalian Selenoprotein O: A Redox-Active Mitochondrial Protein. PLoS ONE, 9(4), e95518.

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Cell Lysis and Chromatin Fractionation

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Cells were lysed with NP40 buffer (1% NP40, 300 mM NaCl, 0.1 mM EDTA, 50 mM Tris pH 7.5) supplemented with protease inhibitor cocktail (Roche). Cell lysate was resolved by gel electrophoresis using NuPAGE Novex gels (Invitrogen), and proteins were transferred onto nitrocellulose membranes, sequentially incubated with primary and secondary antibodies and detected by chemiluminescence. For chromatin fractionation, cell pellets obtained above were resuspended in MNase buffer (1% NP40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 7.5, 0.5% NP40, 10% Glycerol, 2mM CaCl₂) with MNase (Roche) at a final concentration of 3 U/ul and incubated at 37 °C with shaking for 15 minutes. An equal volume of 2X HA-IP buffer (1% NP40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris pH 7.5, 0.05% NP40, 10% Glycerol) was added and tubes were incubated on ice for 15 minutes. The supernatant was collected after spinning at maximum speed, and chromatin-bound proteins were precipitated using 50% TCA (trichloroacetic acid) and resuspended in NP40 buffer. Fractions were probed with different antibodies using gel electrophoresis.

Clairmont C.S., Sarangi P., Ponnienselvan K., Galli L.D., Csete I., Moreau L., Adelmant G., Chowdhury D., Marto J.A, & D’Andrea A.D. (2020). TRIP13 Regulates DNA Repair Pathway Choice through REV7 Conformational Change. Nature cell biology, 22(1), 87-96.

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Western Blot Analysis of TRAIL and DR5

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30 μg of total protein lysate per sample was separated by electrophoresis using 10% Bis-Tris NuPAGE® Novex gels (Invitrogen) and XCell SureLock® Mini-Cell (Invitrogen). Proteins were transferred to polyvinylidene fluoride membranes using an iBlot® gel transfer device (Invitrogen). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and immunoblotted overnight at 4 °C with anti-TRAIL antibody (R&D Systems) or anti-DR5 antibody (R&D Systems) diluted 1:2500 in TBS-T. After washing with TBS-T, membranes were incubated for 1 h with HRP-conjugated anti-goat antibody at room temperature. Membranes were washed with TBS-T and developed with SuperSignal West Pico chemiluminescent substrate (Thermo Life Science). The images were taken with ImageQuant^TM LAS4000 digital imaging system (GE Healthcare).

Kichev A., Rousset C.I., Baburamani A.A., Levison S.W., Wood T.L., Gressens P., Thornton C, & Hagberg H. (2014). Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Signaling and Cell Death in the Immature Central Nervous System after Hypoxia-Ischemia and Inflammation. The Journal of Biological Chemistry, 289(13), 9430-9439.

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Membrane Fractionation and Western Blotting of CB1 Receptors

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Tissue from cultures was harvested for cell fractionation either immediately following treatment or after a 4 day withdrawal. Prior to scraping, cultures were washed three times to remove excess media in an ice-cold HEPES-buffered sucrose solution containing (in mM): 200 sucrose, 1.9 KCl, 6 MgCl₂, 0.5 CaCl₂, 10 glucose, 0.4 ascorbic acid, 25 HEPES, pH 7.3 with KOH.
A crude membrane fraction was prepared and tissue samples were used for western blotting as previously described (Mulholland and Chandler, 2010 (link)). After separation on NuPage Novex gels (4–12% Bis-Tris; Invitrogen Corp., Carlsbad, CA) protein was transferred to Immobilon-P PVDF membranes (Millipore Corporation, Billerica, MA). Membranes were blocked in 1% non-fat dry milk plus 5% BSA and incubated at 4°C overnight in a 1:500 dilution of the L15 anti-CB1 primary antibody (kindly provided by Dr. Ken Mackie, Indiana University, Bloomington, IN). A horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody was then added and bands were detected using enhanced chemiluminescence (ECL). The band corresponding to the molecular weight of CB1 (≈52 kDa) was quantified using computer-assisted densitometry with ImageJ v1.41 (National Institutes of Health, USA).

Pava M.J, & Woodward J.J. (2014). Chronic ethanol alters network activity and endocannabinoid signaling in the prefrontal cortex. Frontiers in Integrative Neuroscience, 8, 58.

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Western Blot Protein Extraction

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Proteins from cells and tumours were extracted with NP40 Lysis Buffer (Biosource, Invitrogen) supplemented with phosphatase and protease inhibitors. Equal amounts of protein (10–50 µg/lane) were separated on NuPAGE Novex gels (Invitrogen), transferred to nitrocellulose or PVDF membranes and immunoreactive bands were visualized using ECL reagents (GE Healthcare).

Kanthou C., Dachs G.U., Lefley D.V., Steele A.J., Coralli-Foxon C., Harris S., Greco O., Dos Santos S.A., Reyes-Aldasoro C.C., English W.R, & Tozer G.M. (2014). Tumour Cells Expressing Single VEGF Isoforms Display Distinct Growth, Survival and Migration Characteristics. PLoS ONE, 9(8), e104015.

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Immunoblotting Analysis of Tau Phosphorylation

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Tissue was homogenized in 200 µl Tris buffer (pH 7.4) containing 10% sucrose and protease inhibitors (Complete; Roche Diagnostics), sonicated and kept at −80°C until use. Protein amounts were quantified using the BCA assay (Pierce), and samples diluted with lithium dodecyl sulphate buffer (2×) supplemented with reducing agents (Invitrogen) and then separated on 4–12% NuPAGE® Novex gels (Invitrogen). Proteins were transferred to nitrocellulose membranes, which were then saturated with 5% non-fat dried milk or 5% bovine serum albumin in TNT (Tris 15 mM pH 8, NaCl 140 mM, 0.05% Tween) and incubated at 4°C for 24 h with the primary antibodies directed against phosphoepitope of tau protein as follows: phospho(p)-Tyr18, pThr212/pSer214 (AT100) (Pierce), pSer262, pSer404, pSer422 (Invitrogen); dephosphorylated tau (Tau1, Millipore), total tau Cter (in-house antibody recognizing the 11 amino acids in COOH-terminal part of tau), HT7 (MN1000, Thermo Scientific). We also used anti-Arc (SantaCruz, sc17839) and anti 14-3-3 (Santa Cruz, 1019) antibodies. Appropriate HRP-conjugated secondary antibodies were incubated for 1 h at room temperature and signals were visualized using chemoluminescence kits (ECL, Amersham Bioscience) and a LAS4000 imaging system (Fujifilm). Results were normalized to actin and quantifications were performed using ImageJ software (Scion Software).

Laurent C., Dorothée G., Hunot S., Martin E., Monnet Y., Duchamp M., Dong Y., Légeron F.P., Leboucher A., Burnouf S., Faivre E., Carvalho K., Caillierez R., Zommer N., Demeyer D., Jouy N., Sazdovitch V., Schraen-Maschke S., Delarasse C., Buée L, & Blum D. (2016). Hippocampal T cell infiltration promotes neuroinflammation and cognitive decline in a mouse model of tauopathy. Brain, 140(1), 184-200.

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Protein-Lipid Vesicle Binding Assay

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MLVs (5 mg/mL), composed of specific molar ratio of selected lipids, were incubated with RahU at 10:1 (w/w) ratio in Tris (20 mM Tris–HCl, 140 mM NaCl, 1 mM EDTA, pH 8.0) or in Hepes (10 mM HEPES, 150 mM NaCl, pH 7.4) buffer at room temperature for 30 min on a rotary shaker at 600 rpm/min. Vesicle-bound RahU was separated from the unbound protein by centrifugation at 60.000 × g and 4°C for an hour. The unbound RahU was precipitated with 100% trichloroacetic acid for 10 min on ice and collected by centrifugation (16.100 × g, 10 min, 4°C). The sediments were washed twice with ice-cold acetone and left to dry completely. Samples were resuspended in NuPAGE LDS loading sample buffer, heated to 100°C for 5 min, proteins resolved on 4–12% NuPAGE Novex gels (Invitrogen, Thermo Fischer Sientific, USA) and visualized by Coomassie Blue staining.

Kočar E., Lenarčič T., Hodnik V., Panevska A., Huang Y., Bajc G., Kostanjšek R., Naren A.P., Maček P., Anderluh G., Sepčić K., Podobnik M, & Butala M. (2021). Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine. Scientific Reports, 11, 6572.

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Western Blot Analysis of Protein Samples

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For all biochemical experiments, tissue was homogenized in 200 μL Tris buffer (pH 7.4) containing 10% sucrose and protease inhibitors (Complete; Roche Diagnostics GmbH), sonicated, and kept at −80°C until use. Protein amounts were evaluated using the BCA assay (Pierce). Protein amounts were evaluated using the BCA assay (Pierce), subsequently diluted with LDS 2X supplemented with reducing agents (Invitrogen) and then separated on 4%–12% NuPage Novex gels (Invitrogen). Proteins were transferred to nitrocellulose membranes, saturated (5% non-fat dry milk or 5% BSA) in TNT (Tris 15 mM pH 8, NaCl 140 mM, 0.05% Tween) and incubated with primary (see Table 1) overnight and then corresponding secondary antibodies (peroxidase labeled horse anti-rabbit 1/5000 or anti-mouse 1/50,000, Vector Laboratories). Immunoreactivity was visualized using chemiluminescence kits (ECL^TM, Amersham Bioscience) and a LAS3000 imaging system (Fujifilm). Results were normalized to GAPDH and quantifications were performed using ImageJ software (Scion Software).

Faivre E., Coelho J.E., Zornbach K., Malik E., Baqi Y., Schneider M., Cellai L., Carvalho K., Sebda S., Figeac M., Eddarkaoui S., Caillierez R., Chern Y., Heneka M., Sergeant N., Müller C.E., Halle A., Buée L., Lopes L.V, & Blum D. (2018). Beneficial Effect of a Selective Adenosine A2A Receptor Antagonist in the APPswe/PS1dE9 Mouse Model of Alzheimer’s Disease. Frontiers in Molecular Neuroscience, 11, 235.

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Western Blot Analysis of Tau Protein

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TBS-soluble samples were incubated with reducing 6× Laemmli SDS buffer (Alfa Aesar, J61337) at 95 °C for 10 min for denaturing conditions or 4× NuPAGE sample buffer (Invitrogen NP0007) for non-denaturing conditions and then loaded on 4–12% NuPAGE Novex gels (Invitrogen). Nitrocellulose membranes were used to transfer proteins and blocked with 5% BSA in TBS with 0.01% tween followed by overnight incubation of primary antibodies (HT7 (Thermo Fisher, MN1000; 1:1,000 dilution), anti-Bassoon (Millipore, ABN255; 1:1,000 dilution), MC1 (Peter Davies; 1:1,000 dilution), anti-total tau (DAKO, A0024; 1:5,000 dilution), PHF1 (Peter Davies; 1:1,000 dilution), anti-Actin (Abcam, ab8227; 1:2,000 dilution), pThr231 (Millipore, MAB5450; 1:1,000 dilution) and Vinculin (Sigma, V9131; 1:1,000 dilution)) diluted in the blocking solution. Horseradish peroxidase (HRP) secondary antibodies (goat anti-mouse HRP conjugated (Invitrogen, 626820; 1:5,000 dilution) and goat anti-rabbit HRP conjugated (Invitrogen, 31460; 1:5,000 dilution)) were incubated for 1 h at RT and the proteins were detected with Supersignal West Pico (Thermo Scientific, 34580) and imaged by using iBright 1500 (Invitrogen). Western blots were analyzed using ImageJ (NIH, v1.53i).

Martinez P., Patel H., You Y., Jury N., Perkins A., Lee-Gosselin A., Taylor X., You Y., Viana Di Prisco G., Huang X., Dutta S., Wijeratne A.B., Redding-Ochoa J., Shahid S.S., Codocedo J.F., Min S., Landreth G.E., Mosley A.L., Wu Y.C., McKinzie D.L., Rochet J.C., Zhang J., Atwood B.K., Troncoso J, & Lasagna-Reeves C.A. (2022). Bassoon contributes to tau-seed propagation and neurotoxicity. Nature Neuroscience, 25(12), 1597-1607.

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