Chloroplast and thylakoid membranes were isolated as described in our previous work (Kim et al. 2012 (link)) and BN-PAGE was conducted as described by Fu et al. (2007) (link). All steps of the sample preparation were carried out at 4°C. Thylakoid membranes were solubilized with mild detergent dodecyl maltoside (Sigma) for 10 min on ice to a final concentration of 1%, and the soluble fraction was transferred to a new tube after high speed centrifugation at 16 000 g for 30 min. The fractions were separated on 5–13.5% native PAGE, with sample buffer (0.05% Serva G, 3% sucrose, 100 mM Bis-Tris–HCl and 0.5 M 6-amino-caproic acid, pH 7.0) by electrophoresis at constant voltage (100 V, for 2 h) in a cold chamber. After BN-PAGE, a lane of the one-dimension (1-D) BN-PAGE gel was cut, and incubated for 20 min in 1× SDS-PAGE sample buffer for denaturation. The sliced 1-D gel was put on a 2-D, 12% SDS-PAGE for electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Scientific, Waltham, MA). For immunoblot analysis, thylakoid proteins were loaded on equal chlorophyll basis (20 µg). Photosystem proteins were detected with Super Signal West Pico Chemiluminescence substrate (Thermo Scientific).
Dodecyl maltoside
Manufactured by Merck Group
Sourced in United States
Dodecyl maltoside is a non-ionic detergent commonly used in the solubilization and purification of membrane proteins. It is a mild detergent that helps maintain the native structure and function of proteins. Dodecyl maltoside is widely used in biochemical and biophysical applications.
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Lab products found in correlation
Cumene hydroperoxide, by Merck Group (5 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Thermo Fisher Scientific (5 mentions)
Coenzyme q2, by Merck Group (5 mentions)
Antimycin a, by Merck Group (5 mentions)
Cytochrome c, by Merck Group (5 mentions)
Glutathione reductase, by Merck Group (4 mentions)
Nadph, by Merck Group (4 mentions)
Erythromycin, by Merck Group (4 mentions)
Aspirin, by Merck Group (3 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (3 mentions)
Atp bioluminescent somatic cell assay kit, by Merck Group (3 mentions)
Apoptosis detection kits for flow cytometry, by BD (3 mentions)
Resorufin, by Merck Group (3 mentions)
7 ethoxyresorufin, by Merck Group (3 mentions)
Nf κb p65, by Santa Cruz Biotechnology (2 mentions)
N nitrosodimethylamine, by Merck Group (2 mentions)
Il6 and tnf α measurement kits, by BD (2 mentions)
N acetyl cysteine (nac), by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (2 mentions)
10 protocols using dodecyl maltoside
1
Chloroplast and Thylakoid Membrane Isolation and BN-PAGE Analysis
2
Cell-free Synthesis of Proteoliposomes
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Cell-free syntheses were performed using the WEPRO7240H Expression kit (Cell Free Sciences) according to protocol described previously (Li et al., 2017 (link)), with slight modifications. In brief, 2 µg purified plasmid DNA was transcribed at 37°C for 6 h. Translation reactions were then performed in the presence of 4 mg/ml soybean liposomes (Avanti Polar Lipids) at 25°C for 20 h. The proteoliposomes were solubilized by 1% m/v dodecylmaltoside (Sigma) and subjected to native PAGE electrophoresis.
3
Hepatoprotective Mechanisms of Natural Compounds
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Aspirin, LPS, NAC, NADPH, reduced glutathione (GSH), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 5,5’-dithio bis-2-nitrobenzoic acid (DTNB), cytochrome c, coenzyme Q2, antimycin A, dodecyl maltoside, N-nitrosodimethylamine (NDMA), erythromycin and ATP bioluminescent somatic cell assay kits were purchased from Sigma (St Louis, MO, USA). 2,7-Dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Kits for mitochondrial membrane potential assays were procured from R & D Systems, MN, USA. Apoptosis detection kits for flow cytometry and IL6 and TNF-α measurement kits were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Polyclonal antibodies against beta-actin, HO-1, IκB-α, NF-κBp65, PARP, Nrf-2 and cytochrome c were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Reagents for electrophoresis and Western blot analyses were purchased from Bio-Rad Laboratories (Richmond, CA, USA).
4
Streptozotocin-Induced Diabetes Assay
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Streptozotocin (STZ), reduced and oxidized glutathione (GSH/GSSG), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NADH, NADPH, cytochrome c, coenzyme Q2, sodium succinate, antimycin A, dodecyl maltoside, resorufin, 7-ethoxyresorufin, methoxyresorufin, Hoechst 33342, and ATP bioluminescent somatic cell assay kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorofluorescein diacetate (DCFDA) was procured from molecular probes (Eugene, OR, USA). Kits for nitric oxide and caspase-3 and caspase-9 assays were purchased from R&D Systems Inc., MN, USA, and that for lipid peroxidation (LPO) from Oxis International Inc. (CA, USA). Kits for GSH/GSSG assay were procured from Promega Corp. (Madison, WI, USA). Apoptosis detection kits for flow cytometry were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). Rin-5F cells were obtained from American Type Culture Collection (Manassas, VA, USA). Polyclonal antibodies against beta-actin, caspase-3, PARP, NOS-2, Nrf2, GLUT 2, Bax, Bcl-2, Akt, and p-Akt were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Reagents for cell culture, SDS-PAGE, and Western blot analyses were purchased from Gibco BRL (Grand Island, NY, USA) and Bio-Rad Laboratories (Richmond, CA, USA).
5
Antioxidant Enzyme Assays Protocol
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Aspirin (acetylsalicylic acid, ASA), reduced glutathione (GSH), oxidized glutathione (GSSG), 5,5′-dithio-bis (2-nitrobenzoic acid), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, dimethyl nitrosamine (DMNA), erythromycin, glutathione reductase, NADH, NADPH, coenzyme Q2, antimycin A, dodecyl maltoside, sodium succinate, cytochrome c, lucigenin and ATP Bioluminescent cell assay kits were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO, USA). 2′,7′-dichlorofluorescein diacetate (DCFDA) was procured from Molecular Probes (Eugene, OR, USA). Kits for SOD and GDH were procured from Abcam (Cambridge, UK), and lipid peroxidation (LPO) kits were obtained from Oxis Int. Inc. (Portland, OR, USA).
6
Hepatic Oxidative Stress Pathway Evaluation
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Cytochrome c, reduced glutathione (GSH), oxidized glutathione (GSSG), 5,5′‐dithio‐bis(2‐nitrobenzoic acid), cumene hydroperoxide, dimethylnitrosamine (DMNA), erythromycin, 7‐ethoxyresorufin, methoxyresorufin, resorufin, dinitrophenylhydrazine (DNPH), lucigenin, glutathione reductase, thiobarbituric acid, NADH, NADPH, coenzyme Q2, sodium succinate, antimycin A, rotenone, dodecyl maltoside, ATP Bioluminescent cell assay kits, and Hexokinase assay kits were purchased from Sigma‐Aldrich Fine Chemicals (St Louis, MO). 2′, 7′‐Dichlorofluorescein diacetate (DCFDA) was procured from Molecular Probes (Eugene, OR). Kits for SOD assay were procured from R & D Systems (Minneapolis, MN) and for glutamate dehydrogenase assay from Abcam (Cambridge, UK).Polyclonal antibodies against CYP2E1, Glut 2, NF‐kB p65, and β‐actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), whereas those against PPAR‐γ and HO‐1 were purchased from Abcam. Reagents for SDS‐PAGE and Western blot analyses were purchased from Gibco BRL (Grand Island, NY) and Bio Rad Laboratories (Richmond, CA).
7
HPLC-Based Protein Analysis Protocol
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HPLC solvents including acetonitrile and water were obtained from Burdick & Jackson (Muskegon, MI). Reagents for protein chemistry including iodoacetamide, dithiothreitol (DTT), ammonium bicarbonate, formic acid, trifluoroacetic acid, acetic acid, dichloroacetic acid (DCA), dodecyl-maltoside, and urea were purchased from Sigma Aldrich (St. Louis, MO). All protein standards were >95% purity. Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Thermo (Rockford, IL), and HLB Oasis SPE cartridges were purchased from Waters (Milford, MA). Proteomics grade trypsin was from Promega (Madison WI). Trypsin-predigested beta-galactosidase (a quality control standard) was purchased from SCIEX (Foster City, CA).
8
Oxidative Stress and Inflammatory Response
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Aspirin, LPS, NAC, malondialdehyde, NADPH, 2-thiobarbituric acid, reduced glutathione (GSH), 1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, 5,5′-dithio bis-2-nitrobenzoic acid (DTNB), cytochrome c, coenzyme Q2, antimycin A, dodecyl maltoside, N-nitrosodimethylamine (NDMA), erythromycin, 7-ethoxyresorufin, resorufin and ATP bioluminescent somatic cell assay kits were purchased from Sigma (St Louis, MO, USA). 2,7-Dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). Kits for mitochondrial membrane potential assays were procured from R & D Systems, MN, USA. Apoptosis detection kits for flow cytometry and IL6 and TNF-α measurement kits were purchased from BD Pharmingen (BD Biosciences, San Jose, USA). Murine macrophage J774.2 cells, which are circulatory monocyte macrophages from tumor bearing mice, were purchased from European Collection of cell cultures (Health Protection Agency Culture Collections, Salisbury, UK). Polyclonal antibodies against beta-actin, HO-1, TNF-α, Nrf-2, IκB-α, cytochrome c, and Bcl-2 and, NF-κBp65 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Reagents for electrophoresis and Western blot analyses were purchased from Bio-Rad Laboratories (Richmond, CA, USA).
9
Biochemical Analysis of Sphingolipid Metabolism
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JTE-013, C6 NBD dihydroceramide (NBD-C6-dhCer) and (2S,3R)-2-ammonio-3-hydroxy-5-((2-oxo-2H-chromen-7-yl)oxy)pentyl hydrogen phosphate were from Cayman Chemical (Ann Arbor, MI). SKi (4-[[4-(4-chlorophenyl)-2-thiazolyl]amino]phenol)40 (link), dithiothreitol (DTT), fatty acid-free bovine serum albumin (BSA), human cytochrome B5 (CYB5, Sigma #C1427, E.C.1.6.2.2), FLAG-agarose beads, FLAG peptide, dodecyl maltoside (DDM), SYPRO-RUBY, Complete© EDTA-free protease inhibitors (CPI), Na3VO4, pyridoxal 5′ phosphate, trifluoracetic acid and acetonitrile were from Merck (Frenchs Forest, NSW, Australia). MG132 was purchased from Selleck Chem (Houston, TX). HPLC columns and vials were purchased from Phenomenex (Torrence, CA, USA).
10
Overexpression and Purification of E. coli ATP Synthase and Quinol Oxidase
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The E. coli F1FO ATP synthase was overexpressed in E. coli strain DK8(ΔuncBEFHAGDC) and purified with the Ni2 + -NTA affinity chromatography as described previously65 (link) in the presence of octyl glucoside (Glycon Biochemicals, D97001-C), Na-deoxycholate (Merck, 302-95-4: 30970), and sodium Na-cholate (Merck, 206986-87-0: C6445). The E. coli bo3 quinol oxidase cloned in plasmid pETcyo was overexpressed in E. coli strain C43(DE3)(ΔcyoABCDE) and purified as described by66 (link) in the presence of dodecyl maltoside (Merck, 69227-93-6: D4641).
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