Nupage 4 12 bis tris sds page

Manufactured by Thermo Fisher Scientific

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The NuPAGE 4%–12% Bis-Tris SDS-PAGE is a pre-cast polyacrylamide gel used for protein separation and analysis. It features a Bis-Tris buffer system and a gradient of 4% to 12% polyacrylamide concentration, allowing for effective separation of a wide range of protein molecular weights.

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NuPAGE 4–12% Bis-Tris SDS-PAGE gel NuPAGE® Novex 4–12% (w/v) Bis-Tris SDS-PAGE gels NuPAGE 4–12% Bis-Tris Gels for SDS-PAGE NuPAGE Bis-Tris SDS–PAGE NuPAGE 4–12% Bis-Tris Bis-Tris SDS-PAGE NuPAGE 12% Bis-Tris NuPage SDS-PAGE NuPAGE Bis-Tris SDS-PAGE

6 protocols using nupage 4 12 bis tris sds page

Western Blot Analysis of Metabolic Enzymes

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Tissue lysates were prepared using TissueLyser II (QIAGEN, Valencia, CA, USA) with T-PER Tissue Protein Extraction Reagent and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Total protein concentration was measured by BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA), and equal protein concentrations were resolved by NuPAGE 4%–12% Bis-Tris SDS-PAGE (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoresed proteins were transferred to nitrocellulose membranes using the iBlot Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA) and blocked with Odyssey Blocking Buffer (PBS) (Li-Cor Biosciences, Lincoln, NE, USA). Membranes were then incubated with rabbit anti-GO, rabbit anti-LDHA (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-HYPDH (Abcam, Cambridge, MA, USA), or rabbit anti-AGT (Abcam, Cambridge, MA, USA) and with mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibody (Abcam, Cambridge, MA, USA). Anti-rabbit IRDye 680 and anti-mouse IRDye 800 secondary antibodies (Li-Cor Biosciences, Lincoln, NE, USA) were used for detection, and signal intensity was measured using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).

Lai C., Pursell N., Gierut J., Saxena U., Zhou W., Dills M., Diwanji R., Dutta C., Koser M., Nazef N., Storr R., Kim B., Martin-Higueras C., Salido E., Wang W., Abrams M., Dudek H, & Brown B.D. (2018). Specific Inhibition of Hepatic Lactate Dehydrogenase Reduces Oxalate Production in Mouse Models of Primary Hyperoxaluria. Molecular Therapy, 26(8), 1983-1995.

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Autophagy Markers in Oligodendrocytes

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The autophagy markers were analyzed in oligodendrocytes 12 and 24 h after treatment with 1.5 mg/mL fibrinogen or 0.1 mg/mL fibrin, with or without mTOR activator MHY1485 (#500554, Calbiochem) or autophagy inhibitor VII (#534360, Calbiochem). Cells were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 1.0% NP-40, 0.5% sodium deoxycholate and Roche protease inhibitor cocktail). Samples were then subjected to NuPAGE 4-12% bis-tris-SDS-PAGE (ThermoFisher) and transferred to a nitrocellulose membrane. Membranes were blocked with SuperBlock (ThermoFisher), incubated with anti-p62 (Cell Signaling, #5114; 1:1,000) or anti-LC3 (Cell Signaling, #4108; 1:1,000) rabbit polyclonal antibodies, and then incubated with HRP-conjugated donkey anti-rabbit secondary antibody (ThermoFisher, #A16023; 1:5,000). Membranes were then treated with SuperSignal™ West Pico PLUS chemiluminescent substrate (#34580, ThermoFisher), exposed to CL-XPosure film (#34097, Thermo Scientific) and developed in a X-OMAT 3000 RA film processor (Kodak). Relative abundance of the LC3-II/I ratio was quantified against the loading control β-actin as described^{79 (link)}.

Montagne A., Nikolakopoulou A.M., Zhao Z., Sagare A.P., Si G., Lazic D., Barnes S.R., Daianu M., Ramanathan A., Go A., Lawson E.J., Wang Y., Mack W.J., Thompson P.M., Schneider J.A., Varkey J., Langen R., Mullins E., Jacobs R.E, & Zlokovic B.V. (2018). Pericyte degeneration causes white matter dysfunction in the mouse CNS. Nature medicine, 24(3), 326-337.

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Western Blotting for Glycogen Synthase

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Tissue lysates were prepared using TissueLyser II (QIAGEN, Valencia, CA) with T-PER Tissue Protein Extraction Reagent and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Total protein concentration was measured by BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA), and equal protein concentrations were resolved by NuPAGE 4%–12% Bis-Tris SDS-PAGE (Thermo Fisher Scientific, Waltham, MA). Electrophoresed proteins were transferred to nitrocellulose membranes using the iBlot Dry Blotting System (Thermo Fisher Scientific, Waltham, MA) and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE). Membranes were then incubated with rabbit anti-glycogen synthase antibody (Cell Signaling Technology, Danvers, MA) and with mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibody (Abcam, Cambridge, MA). Anti-rabbit IRDye 680 and anti-mouse IRDye 800 secondary antibodies (Li-Cor Biosciences, Lincoln, NE) were used for detection, and signal intensity was measured using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE).

Pursell N., Gierut J., Zhou W., Dills M., Diwanji R., Gjorgjieva M., Saxena U., Yang J.S., Shah A., Venkat N., Storr R., Kim B., Wang W., Abrams M., Raffin M., Mithieux G., Rajas F., Dudek H., Brown B.D, & Lai C. (2018). Inhibition of Glycogen Synthase II with RNAi Prevents Liver Injury in Mouse Models of Glycogen Storage Diseases. Molecular Therapy, 26(7), 1771-1782.

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Probing Ribosome-SLFN14 Interactions

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SLFN14(WT)-65 kDa or SLFN14(K218E)-65 kDa proteins in the form of eluates after Ni-NTA resin were mixed with 50 pmol 80S ribosomes, reconstituted from purified 40S and 60S ribosomal subunits, in the presence of 1 mM AMPPNP. The reaction mixture was subjected to centrifugation through 10%–30% SDG prepared in buffer C (20 mM Tris-HCl at pH 7.5, 100 mM KCl, 2.5 mM MgCl₂, 1 mM DTT) in a Beckman SW55 rotor at 53,000 rpm for 75 min at 4°C. The 80S ribosomal peak was assayed by NuPAGE 4%–12% Bis-Tris SDS-PAGE (Invitrogen) and SimplyBlue SafeStain (Invitrogen) staining.

Fletcher S.J., Pisareva V.P., Khan A.O., Tcherepanov A., Morgan N.V, & Pisarev A.V. (2018). Role of the novel endoribonuclease SLFN14 and its disease-causing mutations in ribosomal degradation. RNA, 24(7), 939-949.

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SLFN14 Ribosome Binding Dynamics

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Different forms of SLFN14 were incubated with 40S
subunits, 60S subunits, or 80S ribosomes in the presence or absence
of AMPPNP or ADP and subjected to centrifugation through 10–30%
SDG. Fractions that corresponded to ribosomal complexes were resolved
via NuPAGE 4–12% Bis-Tris SDS–PAGE (Invitrogen) and
stained with SimplyBlue
SafeStain (Invitrogen) or tested with anti-SLFN14 antibodies, as indicated.

Pisareva V.P., Muslimov I.A., Tcherepanov A, & Pisarev A.V. (2015). Characterization of Novel Ribosome-Associated Endoribonuclease SLFN14 from Rabbit Reticulocytes. Biochemistry, 54(21), 3286-3301.

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Affinity Purification of Viral Glycoproteins

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BSR-T7/5 cells grown to 60% confluency were transfected with S-tagged HeV, CedV or MojV F or G constructs or with the empty vector using Lipofectamine™ LTX (Invitrogen, Carlsbad, CA, USA) at a 1:2.1 ratio (µg DNA:µL LTX). After 48 h, lysates were collected in RIPA buffer (Thermo Fisher Scientific) with 1x cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and incubated with S protein agarose (EMD Biosciences Inc., Madison, WI, USA) at 4 °C overnight. Washed beads were boiled in reducing sample buffer (2x LDS NuPage^® sample buffer (Invitrogen), 5% β-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA)). Proteins were resolved by NuPage^® 4–12% Bis-Tris SDS-PAGE (Invitrogen), and detected by Western blot with a horseradish peroxidase (HRP) conjugated anti-S polyclonal rabbit antibody (1:12,500) (Southern Biotechnology, Birmingham, AL, USA).

Cheliout Da Silva S., Yan L., Dang H.V., Xu K., Epstein J.H., Veesler D, & Broder C.C. (2021). Functional Analysis of the Fusion and Attachment Glycoproteins of Mojiang Henipavirus. Viruses, 13(3), 517.

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