Nupage 4 to 12 gels

Manufactured by Thermo Fisher Scientific

Sourced in United States

NuPAGE 4 to 12% gels are pre-cast polyacrylamide gel electrophoresis (PAGE) products designed for the separation and analysis of proteins. They provide a consistent and reliable platform for performing gel electrophoresis experiments.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ecl detection system, by Merck Group (1 mentions) Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions) P44 42 mapk, by Cell Signaling Technology (1 mentions) Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Protein assay, by Bio-Rad (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Bca assay kit, by Thermo Fisher Scientific (1 mentions) Protein a g bead, by Thermo Fisher Scientific (1 mentions) Alexa fluor plus 680, by Thermo Fisher Scientific (1 mentions) Alexa fluor plus 800, by Thermo Fisher Scientific (1 mentions) Sars cov 2 s1, by Sino Biological (1 mentions) Empiria studio software, by LI COR (1 mentions) β tubulin, by Proteintech (1 mentions) Gapdh, by ProSci (1 mentions) Odyssey clx, by LI COR (1 mentions) Sars cov 2 s2, by Sino Biological (1 mentions) Propionyllysine, by PTM Biolabs (1 mentions) Succinyl lysine, by PTM Biolabs (1 mentions)

5 protocols using nupage 4 to 12 gels

Western Blot Analysis of Signaling Pathways

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Protein lysates from cells were prepared and quantified by Bio-Rad protein assay (Bio-Rad). Proteins (30 μg) were loaded to each lane and separated by electrophoresis on NuPAGE 4 to 12% gels (Thermo Fisher Scientific), followed by transferring to 0.2μm nitrocellulose membrane. Membranes were incubated with 5% nonfat dry milk for 1 hour, washed three times for 5 minutes, and incubated with primary antibodies against phospho-JAK1(Tyr1034/1035) (D7N4Z, 1:1000), JAK1 (D1T6W, 1:1000), phospho-STAT5 (Tyr694) (D47E7, 1:1000), STAT5 (D3N2B, 1:1000), phospho-AKT (Ser473) (D9E, 1:1000), AKT (pan) (C67E7, 1:1000), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E, 1:1000), p44/42 MAPK (Erk1/2) (137F5, 1:1000), β-Actin (8H10D10, 1:1000), (Cell Signaling) overnight at 4 °C. Membranes were washed three times for 5 minutes at room temperature and incubated with horseradish-linked secondary antibodies: anti-mouse IgG, HRP-linked antibody (#7076, 1:3000, Cell Signaling Technology), anti-rabbit IgG, HRP-linked antibody (#7074, 1:3000, Cell Signaling Technology) for 1 hour at room temperature followed by another wash. Bands were visualized using an ECL detection system (Millipore).

Zhang Q., Hresko M.E., Picton L.K., Su L., Hollander M.J., Nunez-Cruz S., Zhang Z., Assenmacher C.A., Sockolosky J.T., Garcia K.C, & Milone M.C. (2021). A human orthogonal IL-2 and IL-2Rβ system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia. Science translational medicine, 13(625), eabg6986.

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Protein Expression Analysis Workflow

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Cells were lysed with RIPA lysis buffer (AOqing Biotechnology, Beijing, China) containing ProtLytic Protease Inhibitor Cocktail (New Cell & Molecular Biotech), and the protein concentration was determined with a BCA assay kit (Pierce Biotechnology, USA). The cell lysate was fractionated in NuPAGE™ 4 to 12% gels (Thermo Fisher Scientific) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked and incubated with primary antibodies, including antibodies against LEFTY1, AOC1, GPX3, CRABP2, WFDC2, and S100A2, and then, the commensurate secondary antibody. The bands were visualized with enhanced chemiluminescence following the manufacturer’s instructions (Peirce).

Guo D., Zhang S., Gao Y., Shi J., Wang X., Zhang Z., Zhang Y., Wang Y., Zhao K., Li M., Wang A., Wang P., Gou Y., Zhang M., Liu M., Zhang Y., Chen R., Sun J., Wang S., Wu X., Liang Z., Chen J, & Lang J. (2023). Exploring the cellular and molecular differences between ovarian clear cell carcinoma and high-grade serous carcinoma using single-cell RNA sequencing and GEO gene expression signatures. Cell & Bioscience, 13, 139.

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Immunoprecipitation of PRAME in DLBCL Cells

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DLBCL cells (SU-DHL-4, DB, and OCI-LY10) were lysed using lysis buffer (10 mM Tris-HCl pH8, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40) and nuclei were collected on ice. CHAPS buffer (FIVEphoton Biochemicals) was used for nuclear extraction, and the lysate was incubated with 2 μg of PRAME (Abcam, EPR20330) antibodies overnight at 4°C on a rocker. Protein A/G beads (Thermo Fisher Scientific) were added (20 μL), and samples were rotated for 1 hour at 4°C. Beads were collected by centrifugation and extensively washed with CHAPS lysis buffer. Proteins were eluted into SDS gel loading buffer by heating at 65°C. Proteins were separated using NuPAGE 4% to 12% gels (Thermo Fisher Scientific), transferred to PVDF membranes, and stained as described in EZH2i treatment assay, Western blotting, and Pr20 binding assay.

Takata K., Chong L.C., Ennishi D., Aoki T., Li M.Y., Thakur A., Healy S., Viganò E., Dao T., Kwon D., Duns G., Nielsen J.S., Ben-Neriah S., Tse E., Hung S.S., Boyle M., Mun S.S., Bourne C.M., Woolcock B., Telenius A., Kishida M., Rai S., Zhang A.W., Bashashati A., Saberi S., D’Antonio G., Nelson B.H., Shah S.P., Hoodless P.A., Melnick A.M., Gascoyne R.D., Connors J.M., Scheinberg D.A., Béguelin W., Scott D.W, & Steidl C. (2022). Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma. The Journal of Clinical Investigation, 132(10), e145343.

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Quantitative SARS-CoV-2 Protein Analysis

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After electrophoresis in NuPAGE 4 to 12% gels (Invitrogen), proteins were transferred to nitrocellulose membranes which were processed with the following antibodies: SARS-CoV-2 S1 (no. 40591-T62, Sino Biological), SARS-CoV-2 S2 (no. 40590-T62, Sino Biological), SARS-CoV-2 nucleoprotein (no. 35-579, ProSci), SARS-CoV-2 nsp1 (no. 10-500, ProSci), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; no. 66004-1-Ig, Proteintech), and β-tubulin (no. 66240-1-Ig, Proteintech). Secondary antibodies labeled with infrared dyes were obtained from Thermo Fisher Scientific: donkey anti-mouse, Alexa Fluor Plus 800 (no. A32789); donkey anti-mouse, Alexa Fluor Plus 680 (no. A32788); and goat anti-rabbit, Alexa Fluor Plus 680 (no. A32734). Membranes were scanned on Odyssey CLX (LI-COR Biosciences) and analyzed in Empiria Studio software (LI-COR).

Frolova E.I., Palchevska O., Lukash T., Dominguez F., Britt W, & Frolov I. (2022). Acquisition of Furin Cleavage Site and Further SARS-CoV-2 Evolution Change the Mechanisms of Viral Entry, Infection Spread, and Cell Signaling. Journal of Virology, 96(15), e00753-22.

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Detecting Post-Translational Modifications

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Cells were harvested and lysed in TRIS‐HCL pH 7.4 with 1% triton X‐100 containing protease inhibitors and deacylase inhibitors (1 μM trichostatin A and 20 mM nicotinamide). Lysates were sonicated 5 times 2 seconds at 40 kHz amplitude on ice. Protein concentrations were determined using Pierce BCA protein assay kit (Thermofisher) and equal protein amounts were loaded on NuPAGE 4% to 12% gels (Invitrogen), transferred to nitrocellulose membrane, blocked in 3% BSA in PBS with 0.1% Tween‐20 at room temperature and incubated overnight with antibodies in the same buffer at 4°C. Primary antibodies used: β‐actin (#A5441, Sigma‐Aldrich), propionyllysine (#201, PTM biolabs), succinyllysine (#401, PTM biolabs), acetyllysine (#9441, Cell Signalling), histone 3 propionyllysine 23 (#613987, Active Motif), histone 3 acetyllysine 23 (#07‐355, Millipore). IR‐dye based secondary antibodies (LICOR) were used to detect antibody signals using Odyssey scanner (LICOR).

Lagerwaard B., Pougovkina O., Bekebrede A.F., te Brinke H., Wanders R.J., Nieuwenhuizen A.G., Keijer J, & de Boer V.C. (2020). Increased protein propionylation contributes to mitochondrial dysfunction in liver cells and fibroblasts, but not in myotubes. Journal of Inherited Metabolic Disease, 44(2), 438-449.

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