Nupage novex 4 12 bis tris gel

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The NuPAGE Novex 4–12% Bis-Tris gels are precast polyacrylamide gels used for protein separation and analysis. They have a gradient of 4% to 12% acrylamide concentration, which allows for the separation of a wide range of protein molecular weights. The gels employ a Bis-Tris buffer system for protein resolution.

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Bis-Tris gels (1248 citations) NuPAGE (743 citations) NuPAGE gels (529 citations) NuPAGE Bis-Tris (95 citations) NuPAGE 4–12% Bis-Tris (72 citations) NuPAGE Novex (59 citations) NuPAGE Novex gel (37 citations) NuPage Novex 4–12% Bis-Tris Mini Gels (21 citations) Novex gels (17 citations) NuPAGE Novex 4-12% Bis-Tris precast gels (16 citations)

322 protocols using nupage novex 4 12 bis tris gel

Immunoprecipitation and Signaling of NKp30

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Protein G beads (Dynabeads 100.07D, Thermo Fisher) were used to immunoprecipitate NKp30. Briefly, YT cells were lysed and incubated with β-1,3-glucan (laminarin) at 4 °C for 1–3 days. The protein G beads were conjugated with antibody against β-1,3-glucan or isotype antibody at room temperature for 10 min. These beads were incubated with the mixture of YT cell lysate with β-1,3-glucan at room temperature for 20 min. The immunoprecipitates (complex of NKp30 with its ligand) were eluted from the beads and resolved in NuPAGE Novex 4–12% Bis-Tris Gel (Thermo Fisher, NP0321BOX).
For SFK signaling studies, YT cells were treated with beads conjugated with or without β-1,3-glucan at 37 °C for 5 min, or for the indicated times with soluble β-1,3-glucan. Cell lysates were collected and resolved in NuPAGE Novex 4–12% Bis-Tris Gel (Thermo Fisher). Proteins in the gel were transferred to a nitrocellulose membrane and blotted for NKp30 with 1C01 or anti-phosphorylated SFK (pSFK, Y416).

Li S.S., Ogbomo H., Mansour M.K., Xiang R.F., Szabo L., Munro F., Mukherjee P., Mariuzza R.A., Amrein M., Vyas J.M., Robbins S.M, & Mody C.H. (2018). Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida. Nature Communications, 9, 751.

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Native and Denatured Protein Electrophoresis

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Proteins were subjected to native gel electrophoresis using NuPAGE Novex 3–8% Tris-acetate gels and Novex Tris-glycine native buffer (Life Technologies). Denaturing gel electrophoresis was performed using NuPAGE Novex 4–12% Bis-Tris gels and NuPAGE Mes SDS running buffer (Life Technologies). Where specified, samples were reduced by treatment with 5% (v/v) β-mercaptoethanol. Gels were stained using Instant Blue stain (Sigma-Aldrich).
For Western blot analysis heparinised human plasma was supplemented with cOmplete protease inhibitor cocktail according to the manufacturer's instructions and diluted to 2 mg/ml in PBS/Az. The plasma was supplemented with 0 or 320 μM NaOCl and left to incubate at ambient room temperature overnight. Following reduction using 5% (v/v) β-mercaptoethanol, the proteins were separated using a NuPAGE Novex 4–12% Bis-Tris gel and transferred to PVDF membrane using an iblot 2 (Life Technologies) according to the manufacturer's instructions. After blocking overnight at 4 °C using 5% (w/v) skim milk powder in PBS, the membrane was incubated with polyclonal goat anti-fibrinogen antibody (Life Technologies; diluted 1:2000 in blocking solution) and then incubated with a superclonal anti-goat IgG-HRP conjugate (Life technologies; diluted 1:5000 in blocking solution). Blots were imaged by enhanced chemiluminescence using a Chemidoc touch imaging system (Bio-Rad).

Mañucat-Tan N., Zeineddine Abdallah R., Kaur H., Saviane D., Wilson M.R, & Wyatt A.R. (2020). Hypochlorite-induced aggregation of fibrinogen underlies a novel antioxidant role in blood plasma. Redox Biology, 40, 101847.

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Analytical Characterization of Antibody Charge Isoforms

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EXAMPLE 6

Analytical Cation Exchange Analysis to characterize and determine proportion of charged isoforms was carried out on a MabPac SCX-10 3 μm, 4×50 mm, column (Thermofisher) at 0.5 ml min⁻¹with MES pH 5.6 buffer using salt gradient elution on an Ultimate 3000 HPLC (Dionex) with detection by UV at 280 nm.

Isoelectric Focusing was carried out using non-equilibrium pH gel electrophoresis using IEF 3-10 precast gels (Serva) run in a XCell Surelock Mini-Cell (Invitrogen) with BIO-RAD Power Pac HV and stained with SimplyBlue SafeStain (Invitrogen)

SDS PAGE was carried out on NuPAGE Novex Bis-Tris 4-12% gels (Invitrogen) run in a XCell Surelock™ Mini-Cell (Invitrogen) with BIO-RAD Power Pac HV, MOPs buffer, and stained with SimplyBlue™ SafeStain (Invitrogen). For analysis samples were diluted with loading buffer, NuPAGE LDS (Invitrogen) and for reducing SDS PAGE additionally reduced with DTT.

For Western Blots sample are first separated by non-reducing SDS PAGE, NuPAGE Novex Bis-Tris 4-12% gels (Invitrogen) run in a XCell Surelock Mini-Cell (Invitrogen) with BIO-RAD Power Pac HV, MOPs buffer, transferred to a PVDF membrane and detected using anti-human Kappa light chain AP; e.g. Sigma, Cat. No. K4377. Bound detection antibody is developed using a AP conjugate kit, Cat. No. 170-6432, Biorad.

US10941207B2. Fusion protein comprising three binding domains to 5T4 and CD3 (2021-03-09). BIOTECNOL LIMITED [GB]. Inventors: Nico Mertens [BE], Philip Jeffrey Cunnah [PT], Ana Rita Da Fonseca Ricardo [PT].

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Dose-Dependent Effects of KPT-185 on Cell Signaling

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HT1080 parental and resistant cells were plated at 375,000 cells/well in 6 well plates and treated with either DMSO (0) or 0.03, 0.1, 0.6, 1, or 3 μM KPT-185 for 24 hours prior to collection by trypsinization. Proteins were extracted from cells in Pierce RIPA buffer (Thermo Scientific) supplemented with phosphatase and protease inhibitors (Roche), quantified by the Pierce BCA Protein Assay Kit (Thermo Scientific), and normalized such that for each sample 10 μg of total protein was loaded per lane. Proteins were separated by loading on Novex NuPAGE 4–12 % Bis-Tris Gels (Life Technologies) and transferring to nitrocellulose with the Novex iBlot Gel Transfer Stacks (Life Technologies). The following primary antibodies were used for immunoblot analysis: XPO1 (Santa Cruz), p53 (Santa Cruz), p21 (Abcam), PARP (Cell Signaling), Caspase 3 (Abcam), cleaved Caspase 3 (Cell Signaling), Mcl-1 (Santa Cruz), p-pRb (Cell Signaling), pRb (Cell Signaling), and β-actin (Santa Cruz). Protein signals were detected with infrared linked species-specific secondary antibodies (LI-COR Biosciences). Western blot images were detected with the ODYSSEY Infrared Imaging System (LI-COR Biosciences).

Crochiere M., Kashyap T., Kalid O., Shechter S., Klebanov B., Senapedis W., Saint-Martin J.R, & Landesman Y. (2015). Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitor of Nuclear Export (SINE) compounds. BMC Cancer, 15, 910.

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Novex NuPAGE Western Blot Protocol

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Western blotting was performed using Novex NuPAGE 4–12% Bis-Tris gels (Life Technologies, Carlsbad, CA, USA). Detection was performed using ECL-Prime or ECL-Advance (GE Healthcare, Buckinghamshire, UK). See Additional file 4 for a description of the antibodies used.

Koizume S., Ito S., Nakamura Y., Yoshihara M., Furuya M., Yamada R., Miyagi E., Hirahara F., Takano Y, & Miyagi Y. (2015). Lipid starvation and hypoxia synergistically activate ICAM1 and multiple genes in an Sp1-dependent manner to promote the growth of ovarian cancer. Molecular Cancer, 14, 77.

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Immunoreactivity Analysis of Ricin Toxin

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Ricin 2 µg, 1 µg, and 0.5 µg protein (loaded per individual well) were separated using one-dimensional SDS-PAGE in precast Novex NuPAGE 4–12% bis-tris gels (Life Technologies, Carlsbad, CA). Broad molecular weight protein standards (10–250 kDa) were used as a size reference (Bio-Rad Laboratories, Hercules, CA). Denatured proteins were visually examined using a silver stain for mass spectrometry kit (Pierce, Rockford, IL). Furthermore to assess immunoreactivity, an unstained duplicate gel consisting of separated ricin was electroblotted using the iBlot dry blotting system (Life Technologies). The protein-immobilized PVDF blot was subsequently immunodetected with an affinity purified primary goat-anti ricinus communis antibody using the anti-goat Westernbreeze chromogenic kit (Life Technologies). Of note, to ensure maximal immunoreactivity, the primary antibody (5 mg/mL; diluted in 1% nonfat dry milk) was incubated over night at room temperature on a rocking platform and the next day, the remaining steps in the Westernbreeze chromogenic kit protocol were completed.

Schieltz D.M., McWilliams L.G., Kuklenyik Z., Prezioso S.M., Carter A.J., Williamson Y.M., McGrath S.C., Morse S.A, & Barr J.R. (2015). Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry. Toxicon : official journal of the International Society on Toxinology, 95, 72-83.

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Immunoreactivity Analysis of Ricin Toxin

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SDS-PAGE Analysis of Tobacco Mosaic Virus

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For the sample preparation, 2 μg of TMV from the original purified solution were diluted to a final volume of 15 μl with NaPB buffer pH 7.4, to which 4 μl of the 4x lithium dodecyl sulfate (LDS) loading dye (Life Technologies) were added. This solution was then denatured for 5 min at 95°C, and analyzed on NOVEX NuPAGE 4–12% Bis-Tris gels (Invitrogen) in 1x morpholinepropanesulfonic acid (MOPS) buffer (ThermoFisher Scientific). SeeBlue Plus2 was as molecular standard. The gel was ran at 200 V/120 mA for 40 min. Gels were stained with Commassie Brilliant Blue R-250 and imaged using an AlphaImager system (Protein Simple).

Monroy-Borrego A.G, & Steinmetz N.F. (2022). Three methods for inoculation of viral vectors into plants. Frontiers in Plant Science, 13, 963756.

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Western Blot Protocol for Protein Quantification

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All antibodies used in this study are listed in S2 Table. Immunoblotting was performed as described [27 (link)]. Briefly, tissues were homogenized in ice-cold RIPA lysis buffer (250mM Tris-HCl, pH 7.4, 750 mM NaCl, 5% Triton X-100, 2.5% sodium deoxycholate, 0.5% sodium dodecyl sulphate (SDS), 100 mM NaF, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl (PMSF) and 1% cocktail protein protease and phosphatase inhibitors (Sigma), pH 8.0). Lysates were centrifuged at 14,000x g for 30 min at 4°C, and supernatants were stored at -80°C until analysis. Lysate protein was resolved by SDS-PAGE (Novex NuPAGE 4–12% Bis-Tris gels, Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore sigma). Phosphorylated and total proteins were identified by immunoblotting using the following primary antibodies: Phospho-S6 Ribosomal Protein (Ser240/244) Antibody #2215 and α-Tubulin (DM1A) Mouse mAb #3873 (Cell Signaling Technologies) at 1:1000 dilution. Immunoblots were evaluated using IRDye® 680RD donkey anti-mouse IgG and 800CW donkey anti-rabbit IgG (LI-COR) at 1:15,000 and scanned on an Odyssey Clx Imaging System (LI-COR). Fluorescent signals were quantified using Image Studio Software (LI-COR).

Kanshana J.S., Mattila P.E., Ewing M.C., Wood A.N., Schoiswohl G., Meyer A.C., Kowalski A., Rosenthal S.L., Gingras S., Kaufman B.A., Lu R., Weeks D.E., McGarvey S.T., Minster R.L., Hawley N.L, & Kershaw E.E. (2021). A murine model of the human CREBRFR457Q obesity-risk variant does not influence energy or glucose homeostasis in response to nutritional stress. PLoS ONE, 16(9), e0251895.

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Proteomic Analysis of Phosphopeptides

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Proteins were electrophoresed into Novex NuPAGE 4–12% Bis-Tris gels (Invitrogen) and visualised with colloidal blue staining kit (Invitrogen). Proteins were excised, reduced/alkylated and in-gel digested using trypsin as described previously [75 (link)]. Peptides for direct MS analysis were desalted using C18 StageTips [76 (link)]. Peptides for phosphopeptide enrichment were extracted from the gel using 3% TFA/30% ACN followed by 100% ACN. Before enrichment approximately 3 mg TiO₂ beads were pre-incubated in 20 μl of 85 mg/ml lactic acid in 80% ACN/0.1%TFA. This pre-incubated bead mixture was added to the peptide mixture and incubated for 1 h at room temperature [77 (link)]. After washing once with 10% ACN/0.1% TFA, and twice with 80% ACN/0.1% TFA, peptides were eluted with 2% ammonium hydroxide in 40% ACN (pH 10.5). Eluate was concentrated to 100 μl and 100 μl of 2% TFA was added and peptides desalted using C18 StageTips.

Zich J., May K., Paraskevopoulos K., Sen O., Syred H.M., van der Sar S., Patel H., Moresco J.J., Sarkeshik A., Yates JR I.I.I., Rappsilber J, & Hardwick K.G. (2016). Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests. PLoS Genetics, 12(2), e1005834.

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