Strains were grown overnight in YPD, washed, and transferred to SC –methionine –cysteine medium (2% glucose) until the exponential phase. Cells were then harvested and washed with lysis buffer [200 mM sorbitol, 20 mM HEPES–KOH (pH 6.8), 1 mM EDTA, 50 mM potassium acetate, and protease inhibitors (Roche)]. Next, the cells were broken with glass beads using a FastPrep machine. The concentration of proteins was determined with the Pierce protein assay (Thermo Fisher Scientific), and 14 µg/well was loaded on an Invitrogen NuPAGE Novex bis-Tris gradient gel (4–12%). For detection of the BiFC constructs, a polyclonal anti-GFP antibody (GeneTex, GTX113617) was used, which recognizes both the N- and C-terminal fragments of the yEmVenus fluorescent protein. Detection of histone H3 (loading control) was done using an anti-histone H3 antibody (Abcam, ab1791). The blots were visualized using a FujiFilm LAS-4000 mini system. Quantification was carried out with ImageJ 1.51d software (National Institutes of Health). The intensity of each band was corrected for the background and normalized to the intensity of its respective loading control band.
Nupage novex bis tris gradient gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE Novex Bis-Tris gradient gels are pre-cast polyacrylamide gels used for the separation and analysis of proteins. They feature a Bis-Tris buffer system and a gradient composition that allows for the effective separation of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000 mini system, by Fujifilm (2 mentions)
Nitrocellulose membrane, by LI COR (2 mentions)
Irdye conjugated secondary antibody, by LI COR (2 mentions)
Ab1791, by Abcam (1 mentions)
Gtx113617, by GeneTex (1 mentions)
Anti pgk1, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Roche (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Mouse anti acetylated α tubulin, by Merck Group (1 mentions)
Rabbit anti hmox 1 antibody, by Cell Signaling Technology (1 mentions)
Mouse anti involucrin, by Thermo Fisher Scientific (1 mentions)
Sigmafast protease inhibitor cocktail, by Merck Group (1 mentions)
Image studio software, by LI COR (1 mentions)
Acetylated α tubulin, by Merck Group (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Hmox1, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
7 protocols using nupage novex bis tris gradient gel
1
Detecting BiFC Protein Interactions
2
Fluconazole Effect on Yeast Proteome
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S. cerevisiae strains were grown in SDglu medium for 24 h at 30°C, with or without 20 μg/ml fluconazole. Cells were washed with lysis buffer (200 mM sorbitol, 20 mM HEPES–KOH [pH 6.8], 1 mM EDTA, 50 mM potassium acetate and protease inhibitors [Roche]), and glass beads were added to break them using a FastPrep machine. The amount of proteins was quantified using the Pierce protein assay (Thermo, Fisher), and 6 μg was loaded per well on an Invitrogen NuPage Novex bis-Tris gradient gel (4% to 12%). We used anti-HA (12013819001; Roche) and anti-Pgk1 (459250; Invitrogen) as loading controls. The blots were visualized using a FujiFilm LAS-4000 mini system and accompanying software.
3
Immunoblotting of Cell Lysates
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Immunoblotting was conducted as described previously [34 (link)]. Briefly, cell lysates extracted using the Pierce M-PER Mammalian Cell Lysis Buffer supplemented with 1% SIGMAFAST™ Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) were denatured, separated on a 4–12% NuPage® Novex® Bis-Tris gradient gel (Life Technologies, Carlsbad, CA, USA) at 200 V for 45 min, and transferred onto a nitrocellulose membrane (LI-COR, Lincoln, NE, USA) at 30 V for 1 h. Proteins were detected by incubating first with primary antibodies (rabbit anti-HMOX-1 antibody, Cell Signaling, Danvers, MA, USA; mouse anti-acetylated α-tubulin, mouse anti-AKR1B10, rabbit-anti-DNAI1, and mouse anti-CDC20B, Sigma–Aldrich; mouse anti-CK13 and goat anti-MGMT, Santa Cruz, Dallas, TX, USA; mouse anti-involucrin, Thermo Fisher Scientific, Waltham, MA, USA; rabbit anti-ADH1C and rabbit anti-FANCD2, Abcam, Cambridge, MA, USA), followed by a 30-min incubation with the IRDye-conjugated secondary antibodies (LI-COR). Images were taken using an Odyssey® CLx Imaging System (LI-COR). Densitometry was conducted using LI-COR Image Studio software.
4
Biomarker Expression Profiling by Immunoblotting
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Expression of key biomarkers was assessed at T1, T5, and PT7 by immunoblotting. Cultures were lysed in a SIGMAFAST™-supplemented M-PER Mammalian Cell Lysis Buffer (Pierce, Waltham, MA, USA). Denatured proteins were separated on a 4–12% Nu-PAGE® Novex® Bis-Tris gradient gel (Life Technologies, Carlsbad, CA, USA) and transferred onto a nitrocellulose membrane (LI-COR, Lincoln, NE, USA) by following the manufacturer’s instructions. Primary antibodies used in this study included HMOX-1 (Cell Signaling Technology, Danvers, MA, USA), NQO1 (Santa Cruz Biotechnology, Dallas, TX, USA), AKR1B10 (Sigma-Aldrich), GCS (Santa Cruz Biotechnology), β-actin (Santa Cruz Biotechnology), acetylated α-tubulin (Sigma-Aldrich), DNAI1 (Sigma-Aldrich), CDC20B (Thermo Fisher Scientific, Waltham, MA, USA), Forkhead-box J1 (FoxJ1) (Santa Cruz Biotechnology), and involucrin (Neomarkers, Fremont, CA, USA). IRDye-conjugated secondary antibodies were obtained from LI-COR (Lincoln, NE, USA). The density of the protein bands was quantified using a LI-COR Odyssey imaging system and Image Studio Software (version 5.0).
5
Spinal Cord Protein Expression Profiling
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Five-millimeter-long segments from CHL1−/− and CHL1+/+ mice spinal cord were dissected at the thoracic level. Samples were mechanically dissociated and lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma). Samples were briefly sonicated, heated at 96°C for 5 min, and centrifuged (20,600× g) at 4°C for 5 min. The supernatants were electrophoretically separated by SDS-PAGE on 4%–12% NuPAGE Novex Bis-Tris gradient gels (Invitrogen) and then transferred to nitrocellulose membranes (Invitrogen). Blots were incubated overnight at 4°C with primary antibodies (Table 1 ) and washed three times in Tris-buffered saline with 0.05% Tween 20 before probing with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at RT. Proteins of interest were then visualized with an enhanced chemiluminescence reagent (GE Healthcare, Piscataway, NJ, USA) and quantified with a laser-scanning densitometer (Canon, Lake Success, NY, USA).
6
Hippocampal Protein Expression Analysis
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The harvested hippocampal tissues were subjected to expression analysis by Western blotting as described previously [37 (link)38 (link)]. Briefly, the tissues were homogenized in lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) with protease inhibitors (aprotinin, pepstatin A, and leupeptin) at mg/mL concentrations for protein extraction. The cell lysate was centrifuged (12,000 g for 10 min at 4℃), the supernatant was removed, and the total protein concentration was determined using a protein assay kit (Bio-Rad). Equal amounts of sample protein (50 µg) were electrophoresed in NuPAGE Novex Bis-Tris gradient gels (Invitrogen). The separated bands were then blotted onto nitrocellulose membranes and incubated with blocking solution (0.1% TBST and 5% non-fat milk) for 2 h, followed by incubation with primary antibodies overnight at 4℃. The membranes were washed three times in TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 60 min. Following five to six washes in TBST, immunoreactive bands were visualized using an ECL detection kit (GE Healthcare, Fairfield, CT, USA) and analyzed using Image J software (NIH Image, Bethesda, MD, USA). Protein expression was normalized relative to the expression of β-actin as an internal standard.
7
Western Blot Analysis of SR-B1 Protein
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Twenty μg of total cell lysate was run on NuPAGE Novex Bis-Tris gradient gels (Invitrogen). Proteins were transferred to nitrocellulose and probed using the following antibodies from Sigma: rabbit anti-SR-B1, 1:750, and mouse anti-β-actin, 1:2000. Blots were developed using a SuperSignal West Femto Kit (Pierce, Scoresby, VIC, Australia) and imaged using a LAS-4000 (Fujifilm, Brookvale, NSW, Australia). Blots were quantified using ImageJ software (NIH).
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