Nativepage novex bis tris gel system

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The NativePAGE Novex Bis-Tris Gel System is a laboratory equipment designed for the separation and analysis of native proteins and protein complexes. It utilizes a native polyacrylamide gel electrophoresis (Native PAGE) technique to preserve the native structure and function of proteins during the separation process.

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Bis-Tris gels (1248 citations) NativePAGE gels (23 citations) Novex system (21 citations) Novex gels (17 citations) NativePAGE system (13 citations) NativePAGE Gel system (4 citations) Novex NativePAGE Bis-Tris system (2 citations)

78 protocols using nativepage novex bis tris gel system

Native PAGE Gel Electrophoresis Protocol

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Running Buffers; Invitrogen NativePAGE Novex Bis-Tris Gel System, Cat. no. BN1001BOX, BN1002BOX, and BN1004BOX

Protein Standard; Invitrogen NativeMARK Unstained Protein Standard, Cat. no. LC0725

3–12% Bis-Tris Gel; NativePAGE 3–12% Bis-Tris Gel, Cat. no. BN1003BOX

Protocol and reagents were adapted from Sancak et al (2013) (link). Gel electrophoresis running buffers were prepared according to the manufacturer’s protocol for the Invitrogen NativePAGE Novex Bis-Tris Gel System. Running buffers were cooled to 4°C before use, and electrophoresis was performed at 4°C. Invitrogen NativeMARK Unstained Protein Standard was used to estimate molecular weight. Gels were run at 40 V for 30 min. Voltage was then increased to 100 V for 1 h, and subsequently to 250 V for 90 min. When the dye front had traveled through ∼1/3 of the gel, electrophoresis was paused, and the Dark Blue Cathode Buffer was replaced with Light Blue Cathode Buffer, as per the manufacturer’s protocol.

MacEwen M.J., Markhard A.L., Bozbeyoglu M., Bradford F., Goldberger O., Mootha V.K, & Sancak Y. (2020). Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU. Life Science Alliance, 3(10), e202000718.

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Blue-Native Polyacrylamide Gel Electrophoresis

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Blue-native-PAGE (BN-PAGE) was carried out using the NativePAGE™ Novex Bis–Tris Gel system (Invitrogen); a precast polyacrylamide system used for the separation of proteins in the non-denatured state. The system is based on the method described by Schagger and von Jagow [49 (link)], which uses Coomassie blue G-250 as the charge-shift molecule. This molecule binds to proteins and confers a net negative charge, while maintaining the proteins in the native state without any denaturation [50 (link)]. It is added to the samples containing non-ionic detergent prior to loading. It is also present in the cathode buffer to provide a continuous flow of Coomassie blue G-250 into the gel. BN-PAGE was performed at 140 V for 2 h using a 4%–12% Bis–Tris gel, according to the manufacturer's instructions. Upon completion of the run, the gels were placed in fix solution (40% v/v methanol and 10% v/v acetic acid) and microwaved at high voltage for 45 s, followed by incubation for 30 min at room temperature on a shaker. The fix solution was discarded and Coomassie Blue stain (0.1% w/v Coomassie Brilliant Blue R-250, 40% v/v methanol and 10% v/v glacial acetic acid) was added to the gel, followed by microwaving and overnight incubation at room temperature, with shaking. The gel was destained with destaining solution (8% v/v acetic acid) and microwaved again until clear enough for imaging.

Seyedmohammad S., Fuentealba N.A., Marriott R.A., Goetze T.A., Edwardson J.M., Barrera N.P, & Venter H. (2016). Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore. Bioscience Reports, 36(2), e00322.

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Characterizing Recombinant EhDHS1/2 Proteins

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Approximately 10 μg of recombinant EhDHS1/2 were electrophoresed on a 4–16% BN-PAGE gel. BN-PAGE was performed using the NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to the manufacturer’s protocol. After electrophoresis, the gel was fixed and destained. An identical gel was also electroblotted onto a polyvinylidene fluoride (PVDF) membrane at 10 V for 2 hr. Following electroblotting, the PVDF membrane was fixed in 10% acetic acid for 20 min, rinsed with deionized water, and subjected to immunoblot analysis as described below.

Jeelani G, & Nozaki T. (2021). Eukaryotic translation initiation factor 5A and its posttranslational modifications play an important role in proliferation and potentially in differentiation of the human enteric protozoan parasite Entamoeba histolytica. PLoS Pathogens, 17(2), e1008909.

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Analyzing Mitochondrial Respiratory Complexes

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BN-PAGE experiments were performed using NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to the manufacturer's recommendations. Heart mitochondria (40 µg) were solubilized with NativePAGE Sample Buffer containing 1% dodecylmaltoside (DDM). After 20 minutes on ice, samples were centrifuged (30 min, 16000×g, 4°C). Supernatants were supplemented with NativePAGE G250 Sample Additive and fractionated through 4–16% NativePAGE Novex Bis-Tris Gel. Respiratory chain complexes were transferred onto PVDF membrane and detected using specific antibodies. Pulse labeling of mitochondrial transcription products was performed in isolated mitochondria according to [38] (link), [39] (link). In organello translation was performed as previously described [22] (link), [40] (link).

Metodiev M.D., Spåhr H., Loguercio Polosa P., Meharg C., Becker C., Altmueller J., Habermann B., Larsson N.G, & Ruzzenente B. (2014). NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly. PLoS Genetics, 10(2), e1004110.

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Protein Analysis by SDS-PAGE and Western Blot

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Samples were subjected to SDS-PAGE under denaturing and non-denaturing conditions using the NativePAGE™ Novex^® Bis-Tris gel system (Invitrogen™) following the manufacturer’s instructions. After Electrophoresis, gels were stained with Coomassie^® Brilliant blue G250 for visualization of proteins or transferred to 0.2 µM PVDF membrane (Roche) by western blotting. Blots were incubated with 0.2 µg/ml mouse monoclonal anti-BPI specific antibody (Santa Cruz Biotech Inc.), 0.01 mM rabbit polyclonal anti-Rac2 specific antibody (Cell Signalling Technology- Rac 1/2/3 antibody) or 1.0 μg/ml of monoclonal anti-actin antibody (Millipore, UK). The secondary antibodies used were HRP-linked rabbit anti-mouse and goat anti-rabbit IgG (Cell Signalling Technology). Immunoreactivity was detected using Immobilon™ Western Chemiluminescent HRP- substrate (Millipore) solution and a G-Box Chemie XL (Syngene) and analyzed using GeneSnap and GeneTools software.

McQuillan K., Gargoum F., Murphy M.P., McElvaney O.J., McElvaney N.G, & Reeves E.P. (2020). Targeting IgG Autoantibodies for Improved Cytotoxicity of Bactericidal Permeability Increasing Protein in Cystic Fibrosis. Frontiers in Pharmacology, 11, 1098.

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BN-PAGE Analysis of Mitochondrial Supercomplexes

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BN-PAGE analyses were performed as previously described [24 (link)] using isolated mitochondria lysed with n-dodecyl-β-D-maltoside using the Native PAGE Novex Bis-Tris Gel system (Invitrogen) according to the manufacturer's instructions. Briefly, 30 μg of isolated mitochondria was solubilized using sodium dodecyl maltoside. Digitonin was included in the lysis buffer for detection of mitochondrial supercomplexes. The suspensions were centrifuged at 20000 × g for 10 min at 4°C, after which proteins in the resulting supernatants were resolved by PAGE on a native polyacrylamide Novex 3–12% Bis-Tris gel (Invitrogen) and then transferred to a polyvinylidene fluoride (PVDF) membrane. After fixing with 8% acetic acid, the membrane was blocked with 5% skim milk in TBS-T (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 h and immunoblotted using Anti-OxPhos Blue Native WB Antibody Cocktail (Invitrogen, 457999).

Han J., Kim S.J., Ryu M.J., Jang Y., Lee M.J., Ju X., Lee Y.L., Cui J., Shong M., Heo J.Y, & Kweon G.R. (2019). Chloramphenicol Mitigates Oxidative Stress by Inhibiting Translation of Mitochondrial Complex I in Dopaminergic Neurons of Toxin-Induced Parkinson's Disease Model. Oxidative Medicine and Cellular Longevity, 2019, 4174803.

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Protein Complex Analysis by Blue Native-PAGE

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Blue native-PAGE was performed using the Native PAGE Novex bis-Tris Gel System (Invitrogen). Five micrograms of protein analytes were mixed with NativePAGE sample buffer (Invitrogen) and NativePAGE 5% G-250 sample additive (Invitrogen). Mixtures were incubated for 30 min on ice. After incubation, analytes were applied to NativePAGE Novex 4–16% bis-Tris gel (Invitrogen), and electrophoresis was performed at 150 V. Preparation of running buffer and other conditions on Blue Native-PAGE were carried out in accordance with the manufacturer’s instructions. NativeMark unstained protein standard (Invitrogen) was used as a molecular weight marker for Blue Native-PAGE. Gels were stained with CBB R-250.

Moriyama T., Ito A., Omote S., Miura Y, & Tsumoto H. (2015). Heat Resistant Characteristics of Major Royal Jelly Protein 1 (MRJP1) Oligomer. PLoS ONE, 10(5), e0119169.

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BN-PAGE Separation and Western Blot Analysis

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The blue-native PAGE (BN-PAGE) was performed using the NativePAGE Novex Bis-Tris gel system (BN1001BOX; Invitrogen) according to the manufacturer's protocol. Briefly, the crude mitochondria were lyzed in 1% DDM containing 1 mM PMSF, 10 mM sodium azide and 10 mM sodium ascorbate. After lyzing on ice for 30 min, the cell lysates were centrifuged at 22,000 g for 30 min. The supernatant was collected and added with Coomassie Blue G250 dye, with a final concentration of 0.2%. Equal amounts of proteins were separated by a 3–12% gradient NativePAGE gel. Proteins were transferred to the PVDF membrane (EMD Millipore). The membrane was then incubated in 8% acetic acid, activated with methanol and blocked with non-fat milk. The diluent primary antibody against Drp1 was incubated overnight at 4°C and detected the following day with an HRP-linked secondary antibody using an ECL reagent.

He X., Wang L., Tsang H.Y., Liu X., Yang X., Pu S., Guo Z., Yang C., Wu Q., Zhou Z., Cen X, & Zhao H. (2024). GTPBP8 modulates mitochondrial fission through a Drp1-dependent process. Journal of Cell Science, 137(8), jcs261612.

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Mitochondrial Protein Analysis by Western Blot

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Samples were lysed in lysis buffer [50 mM tris (pH 7.4), 150 mM KCl, 1
mM EDTA, 1% NP-40, 5 mM NAM, 1 mM sodium butyrate, protease inhibitors].
Proteins were separated by SDS-PAGE and transferred onto nitrocellulose or
polyvinylidene difluoride membranes. Blocking and antibody incubations were
performed in 5% bovine serum albumin. SIRT1 antibody was from Abcam,
anti-FOXO1 antibody was from Cell Signaling, PAR antibody was from Millipore,
and anti–acetyl-FRKH (FOXO) antibody was from Santa Cruz Biotechnology.
Antibody cocktail (the MitoProfile Total OXPHOS Rodent WB Antibody Cocktail) for
mitochondrial subunits was purchased from MitoSciences. Antibody detection
reactions were developed by enhanced chemiluminescence (Advansta) using x-ray
films or imaged using the c300 imaging system (Azure Biosystems). Quantification
was done using ImageJ software. Blue native PAGE on isolated mitochondria from
muscle or C2C12 cells was performed using the NativePAGE Novex Bis-Tris Gel
System (Invitrogen), as described previously (49 (link)).

Ryu D., Zhang H., Ropelle E.R., Sorrentino V., Mázala D.A., Mouchiroud L., Marshall P.L., Campbell M.D., Ali A.S., Knowels G.M., Bellemin S., Iyer S.R., Wang X., Gariani K., Sauve A.A., Cantó C., Conley K.E., Walter L., Lovering R.M., Chin E.R., Jasmin B.J., Marcinek D.J., Menzies K.J, & Auwerx J. (2016). NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation. Science translational medicine, 8(361), 361ra139.

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Quantitative LDH Isoenzyme Analysis

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LDH isoenzyme patterns were determined by previously published methods (Leiblich et al. 2006 (link)). Briefly, the LDH isoenzymes in the subcellular fractions from the cerebral cortex samples were separated on NativePAGE Novex Bis-tris Gel System (Invitrogen; Carlsbad, CA, USA). Regions of LDH activity were made visible by using a staining solution containing: 0.1 M sodium lactate, 1.5 mM NAD, 0.1 M Tris-HCl (pH 8.6), 10 mM NaCl, 5 mM MgCl₂, 0.03 mg/ml phenazinmethosulphate (PMS) and 0.25 mg/ml nitrobluetetrazolium (NBT). The assay relies on the conversion of lactate to pyruvate, with the production of NADH and H⁺. The NADH then reduces PMS, which in turn reduces NBT to give an insoluble product, diformazan. Protein extracted from human heart and skeletal muscles served as positive controls and were obtained from Novus Biologicals. LDH isoenzyme bands were identified by comparison of retention time to standards. Individual bands were quantified by densitometry. Values for individual isoenzymes are expressed as the percent of total isoenzyme (the sum of the densitometry of all isoenzymes in that sample), and reflect the mean ± standard deviation (SD) of three gels.

Duka T., Anderson S.M., Collins Z., Raghanti M.A., Ely J.J., Hof P.R., Wildman D.E., Goodman M., Grossman L.I, & Sherwood C.C. (2014). SYNAPTOSOMAL LACTATE DEHYDROGENASE ISOENZYME COMPOSITION IS SHIFTED TOWARD AEROBIC FORMS IN PRIMATE BRAIN EVOLUTION. Brain, behavior and evolution, 83, 216-230.

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