Nupage precast gel system

Manufactured by Thermo Fisher Scientific

Sourced in United States

The NuPAGE Precast Gel System is a laboratory equipment product designed for protein electrophoresis. It provides pre-cast polyacrylamide gels for the separation and analysis of proteins.

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Lab products found in correlation

Western lightning plus ecl, by PerkinElmer (3 mentions) Pvdf membrane, by GE Healthcare (2 mentions) Ecl prime, by GE Healthcare (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Las 4000 imaging system, by Fujifilm (1 mentions) Polyvinylidene fluoride membrane, by GE Healthcare (1 mentions) Pierce protease inhibitor, by Thermo Fisher Scientific (1 mentions) Mouse anti vinculin, by Merck Group (1 mentions) Rabbit anti actin, by Merck Group (1 mentions) Bradford protein assay, by Bio-Rad (1 mentions) Phosstop, by Roche (1 mentions) Ecl plus reagent, by PerkinElmer (1 mentions) Mab374, by Santa Cruz Biotechnology (1 mentions) 0.45 μm pvdf membrane, by GE Healthcare (1 mentions) Image studio lite, by LI COR (1 mentions) Pierce ha tag co ip kit, by Thermo Fisher Scientific (1 mentions) Flag tag (flag), by Cell Signaling Technology (1 mentions) Irdye 680rd, by LI COR (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Merck Group (1 mentions)

Spelling variants of this product

NuPAGE (743 citations) NuPAGE gels (529 citations) NuPAGE system (144 citations) Precast gels (60 citations) NuPAGE precast gels (38 citations) NuPAGE gel system (22 citations)

8 protocols using nupage precast gel system

Immunoblot Analysis of DNA Repair Proteins

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Samples were homogenized in lysis buffer and sonicated before immunoblot
analysis. 10–20 μg of lysate was run on 4–12%
Bis-Tris acrylamide gels (NuPAGE Precast Gel System, Life Technologies) before
blotting on a PVDF membrane (GE Healthcare). Membranes were incubated with mouse
antibody to Xrcc4 (1:500 dilution; Santa Cruz Biotechnology, sc-365055), rabbit
antibody to Lig4 (1:250 dilution; Santa Cruz Biotechnology, sc-28232), rabbit
antibody to XLF (1:2,000 dilution; Sigma-Aldrich, V9264), mouse antibody to
vinculine (1:2,000 dilution; Sigma-Aldrich, V9264), rabbit antibody to PC1 and
PC3 (1:1,000 dilution; Millipore, AB10553) or rabbit antibody to PC2 (1:200
dilution; Millipore, AB15610). Proteins were visualized using Western Lightning
Plus-ECL (PerkinElmer) and the LAS-4000 imaging system (Fuji). Quantification
was performed using AIDA software (Raytest, version 4.27).

Dooley J., Tian L., Schonefeldt S., Delghingaro-Augusto V., Garcia-Perez J.E., Pasciuto E., Di Marino D., Carr E.J., Oskolkov N., Lyssenko V., Franckaert D., Lagou V., Overbergh L., Vandenbussche J., Allemeersch J., Chabot-Roy G., Dahlstrom J.E., Laybutt D.R., Petrovsky N., Socha L., Gevaert K., Jetten A.M., Lambrechts D., Linterman M.A., Goodnow C.C., Nolan C.J., Lesage S., Schlenner S.M, & Liston A. (2016). Genetic Predisposition for Beta Cell Fragility Underlies Type 1 and Type 2 Diabetes. Nature genetics, 48(5), 519-527.

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Western Blotting of EBV-Transformed Cell Lines

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Cell pellets from EBV clones, generated from fresh PBMCs(4), were solubilized in lysis buffer (50mM Tris-HCl pH 7,5, 100mM NaCl, 5% glycerol, 2mM DTT and 1% Triton-X, protease inhibitor (Pierce) and phosphatase inhibitor (Roche)) and sonicated. Fifty μg of lysate were separated on 4–12% Bis-Tris acrylamide gels (NuPAGE Precast Gel System, Life Technologies) and blotted on a PVDF membrane (GE Healthcare). For nuclear extracts 5μg was loaded. After blotting, membranes were incubated using specific antibodies; r α-IKAROS (Cell Signaling, D10E5), m α-vinculine (Sigma-Aldrich, V9264), m α-histone H1 (Santa Cruz, sc-393358), m α-Flag (Cell Signaling, 9A3), m α-HA (Eurogentec, 16B12), m α-GAPDH (Invitrogen, MA5–15738), Proteins were revealed using western Lightning Plus-ECL (Perkin Elmer) or ECL prime (GE Healthcare). All blots were acquired with the G:box Chemi-XRQ. Quantification was performed using AIDA software (Raytest, version 5.0).

Van Nieuwenhove E., Garcia-Perez J.E., Helsen C., Rodriguez P.D., van Schouwenburg P.A., Dooley J., Schlenner S., van der Burg M., Verhoeyen E., Gijsbers R., Frietze S., Schjerven H., Meyts I., Claessens F., Humblet-Baron S., Wouters C, & Liston A. (2018). A kindred with Mutant IKAROS and autoimmunity.. The Journal of allergy and clinical immunology, 142(2), 699-702.e12.

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Protein Quantification and Immunoblotting

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Fresh PBMCs and primary fibroblasts were solubilized in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM egtazic acid, 1% Triton-X, phosphatase inhibitor (PhosSTOP [Roche, Basel, Switzerland]), and protease inhibitor (Pierce Protease Inhibitor [ThermoFisher Scientific, Waltham, Mass]). A quantity of 50 μg of lysate, which was determined by using a Bradford Protein Assay (Bio-Rad, Hercules, Calif), was separated on a 4% to 12% bis-Tris acrylamide gel with 3-(N-morpholino)propanesulfonic acid buffer (NuPAGE Precast Gel System [Thermo Fisher Scientific]) before blotting on polyvinylidene fluoride membrane (GE Healthcare). After blocking, the membranes were incubated with specific primary antibodies: rabbit anti-Sec61α1 (Abcam, Cambridge, United Kingdom, Ab183046), rabbit anti-Actin (Sigma-Aldrich, St Louis, Mo, A2103), mouse anti–glyceraldehyde-3-phosphate dehydrogenase (Thermo Fisher Scientific, MA5-15738), and mouse anti-Vinculin (Sigma, V9264). Proteins were revealed by using ECL Prime (GE Healthcare, Chicago, Ill) or Western Lightning Plus-ECL (Perkin Elmer, Waltham, Mass) with the G:box Chemi-XRQ and quantified by using ImageJ software.

Van Nieuwenhove E., Barber J.S., Neumann J., Smeets E., Willemsen M., Pasciuto E., Prezzemolo T., Lagou V., Seldeslachts L., Malengier-Devlies B., Metzemaekers M., Haßdenteufel S., Kerstens A., van der Kant R., Rousseau F., Schymkowitz J., Di Marino D., Lang S., Zimmermann R., Schlenner S., Munck S., Proost P., Matthys P., Devalck C., Boeckx N., Claessens F., Wouters C., Humblet-Baron S., Meyts I, & Liston A. (2020). Defective Sec61α1 underlies a novel cause of autosomal dominant severe congenital neutropenia. The Journal of Allergy and Clinical Immunology, 146(5), 1180-1193.

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CYP4F3-POR/cytb5 Co-immunoprecipitation

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Co‐immunoprecipitation (co‐IP) of CYP4F3 and POR/cytb5 from protein extracts of HEK293T cells overexpressing these proteins was performed using the Pierce™ HA‐Tag co‐IP Kit (ThermoFisher Scientific) following manufacturer's protocol. Protein elution from the anti‐HA beads was done by heating for 10 min at 100°C. Proteins from input and immunoprecipitated samples were separated on a 4–12% NuPAGE Precast Gel System (ThermoFisher Scientific) and transferred to a 0.45 μM PVDF membrane (GE Healthcare, #10600023, Chicago, USA). Antibodies against GAPDH (Merck Millipore, MAB374, Burlington, USA), HA (Santa Cruz Biotechnology, sc‐7392, Dallas, USA), or Flag (Cell Signalling Technology, #14793, Danvers, USA) and secondary antibodies goat‐anti‐rabbit and rabbit‐anti‐mouse IgG (Dako, P0448 and P0260, Glostrup, Denmark) were used. Detection was performed with an ImageQuant Las4000 using the western lightning Plus‐ECL reagent (Perkin Elmer, Waltham, USA). Western blots were analysed, and signal intensities were quantified using Image Studio Lite (LI‐COR Biosciences, Lincoln, USA). Immunoprecipitated samples were normalized to the corresponding input samples.

Smeets E., Huang S., Lee X.Y., Van Nieuwenhove E., Helsen C., Handle F., Moris L., El Kharraz S., Eerlings R., Devlies W., Willemsen M., Bücken L., Prezzemolo T., Humblet‐Baron S., Voet A., Rochtus A., Van Schepdael A., de Zegher F, & Claessens F. (2022). A disease‐associated missense mutation in CYP4F3 affects the metabolism of leukotriene B4 via disruption of electron transfer. Journal of Cachexia, Sarcopenia and Muscle, 13(4), 2242-2253.

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Whole Spinal Cord Protein Analysis

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Whole spinal cords were prepared in RIPA buffer (Thermofisher, catalog number: 89900) and resolved using the NuPAGE^® precast gel system (Thermofisher, catalog number: 89900) by SDS-PAGE. Extracts (20 μg) were run on Novex^® Bis-Tris 12% gels and transferred onto an iBlot2 transfer stack nitrocellulose membrane (Thermofisher, catalog number: 89900) using the iBlot2 Dry Blotting system unit (Thermofisher, catalog number: 89900). After protein transfer, the membranes were blocked for 1 h in 5% non-fat dry milk prepared in 1× PBS with 0.1% Tween-20. The membranes were then incubated with the corresponding antibodies overnight (see Table 1: List of antibodies). Thereafter, the membranes were incubated with IRDye 680RD or 800CW secondary antibodies (Li-cor) followed by visualization using a near-infrared imager (Odyssey; Li-cor) (Blanco-Redondo et al., 2019 (link)). Band intensities were analyzed by Fiji ImageJ.

Buettner J.M., Sowoidnich L., Gerstner F., Blanco-Redondo B., Hallermann S, & Simon C.M. (2022). p53-dependent c-Fos expression is a marker but not executor for motor neuron death in spinal muscular atrophy mouse models. Frontiers in Cellular Neuroscience, 16, 1038276.

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SDS-PAGE Separation of PufX Protein

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The NuPAGE pre-cast gel system (Invitrogen, USA) was used to separate polypeptides by SDS-polyacrylamide gel electrophoresis. Purified membranes were incubated at 70 °C for 10 min in NuPAGE LDS sample buffer (Invitrogen, USA). Immunodetection of the PufX protein was performed using enhanced chemiluminescence detection regents supplied by Amersham (ECL Detection System, USA).

Qian P., Martin E.C., Ng I.W, & Hunter C.N. (2017). The C-terminus of PufX plays a key role in dimerisation and assembly of the reaction center light-harvesting 1 complex from Rhodobacter sphaeroides. Biochimica et Biophysica Acta, 1858(9), 795-803.

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SDS-PAGE and Western Blot Analysis

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SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was carried out using a 12 % Nu-PAGE® pre-cast gel system (Invitrogen). Subsequently, the separated proteins were electro-blotted onto a nitrocellulose membrane (Sigma-Aldrich, Saint Louis, MO). After blocking the membrane with 5 % milk in PBS-T, specific proteins were detected with primary antibodies at indicated dilutions followed by goat-anti-rabbit or goat-anti-mouse antibodies conjugated with HRP (Sigma-Aldrich). Cellular α-tubulin, employed as an internal loading control protein, was detected with mouse monoclonal anti-tubulin antibody (1:10,000 dilution) that in turn was detected with (1: 20,000) goat anti mouse-HRP (Sigma-Aldrich). The bound HRP conjugate antibodies were reacted with the WestDura SuperSignal chemiluminescent reagent (Pierce) according to the manufacturer’s instructions and visualized on X-ray film (X-Omat; Kodak, NY).

Rai D.K., Lawrence P., Kloc A., Schafer E, & Rieder E. (2015). Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections. Virology Journal, 12, 224.

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SDS-PAGE Expression Analysis Protocol

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EXAMPLE 3

Expression Analysis Using SDS-PAGE

LDS sample buffer, fourfold concentrate (4×LDS): 4 g glycerol, 0.682 g TRIS (tris-(hydroxymethyl)-aminomethane), 0.666 g TRIS-HCl (tris-(hydroxymethyl)-aminomethane-hydrochloride), 0.8 g LDS (lithium dodecyl sulfate), 0.006 g EDTA (ethylene diamin tetra acid), 0.75 ml of a 1% by weight (w/w) solution of Serva Blue G250 in water, 0.75 ml of a 1% by weight (w/w) solution of phenol red, add water to make a total volume of 10 ml.

The culture broth containing the secreted conjugate was centrifuged to remove cells and cell debris. An aliquot of the clarified supernatant was admixed with 1/4 volumes (v/v) of 4×LDS sample buffer and 1/10 volume (v/v) of 0.5 M 1,4-dithiotreitol (DTT). Then the samples were incubated for 10 min. at 75° C. and protein separated by SDS-PAGE. The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 10% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES running buffer was used. The polypeptides and polypeptide-conjugates could be clearly detected (see FIG. 18).

US10280214B2. Glycosylated repeat-motif-molecule conjugates (2019-05-07). None [DE], None [DE], None [DE]. Inventors: Stephan Fischer [DE], Sabine Imhof-Jung [DE], Erhard Kopetzki [DE].

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