Dihydrolipoamide was synthesized from lipoamide using sodium borohydride as previously described 34 (link), 35 . All PCR primers were purchased from Life Technologies (Carlsbad, CA). ε-amino-N-caproic acid was obtained from MP Biochemicals. Acrylamide/bisacrylamide, ammonium persulfate, Bradford protein assay solution, and Coomassie brilliant blue (CBB) R-250 were from Bio-Rad laboratories (Richmond, CA, USA). NADH, BSA, lipoamide, EDTA, and NBT chloride tablets were obtained from Sigma (St. Louis, MO, USA). Serva Blue G was purchased from Serva (Heidelberg, Germany). Rabbit anti-DLDH polyclonal antibodies (IgG) and goat anti-rabbit IgG conjugated with horseradish peroxidase were purchased from US Biological (Swampscott, MA, USA) and Invitrogen (San Diego, CA, USA), respectively. Hybond-C membrane and an ECL immunochemical detection kit were obtained from GE Healthcare (Piscataway, NJ, USA).
Serva blue g
Manufactured by Serva Electrophoresis
Sourced in Germany
Serva Blue G is a high-quality protein staining dye used in gel electrophoresis. It is a sensitive and reliable stain for the visualization of proteins in polyacrylamide gels.
Automatically generated - may contain errors
Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Bradford protein assay solution, by Bio-Rad (3 mentions)
Goat anti rabbit igg conjugated with horseradish peroxidase, by US Biological (3 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (3 mentions)
Acrylamide bisacrylamide, by Bio-Rad (2 mentions)
Hybond c membrane, by GE Healthcare (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
ε amino n caproic acid, by MP Biomedicals (2 mentions)
Nbt chloride tablets, by Merck Group (2 mentions)
Rabbit anti dldh polyclonal antibodies igg, by US Biological (2 mentions)
Ammonium persulfate, by Bio-Rad (2 mentions)
Lipoamide, by Merck Group (2 mentions)
Hybond, by Cytiva (2 mentions)
Proteinase k, by Merck Group (1 mentions)
Columbia blood agar plates, by bioMérieux (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
N dodecyl β d maltoside ddm, by Merck Group (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Silver nitrate, by Merck Group (1 mentions)
8 protocols using serva blue g
1
Dihydrolipoamide Synthesis and Antibody Generation
2
Bacterial LPS Pattern Comparison
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Lipooligosaccharide (LPS) electrophoresis was used to compare the LPS pattern between throat and corresponding blood isolates and was carried out as described in [56 (link)]. Briefly, bacteria were grown over night on Columbia blood agar plates (bioMérieux). A 1-ml bacterial suspension in phosphate buffered saline (PBS) (1.5 mM KH2PO4, 2.7 mM KCl, 8.5 mM Na2HPO4, 137 mM NaCl, pH 7.4) at an optical density at 600 nm (OD600) of 0.6 was pelleted at 13,000 × g for 1 min, and pellets were taken up in 50 μl lysis buffer (2% SDS, 4% β-mercaptoethanol, 10% glycerol, 1 M Tris [pH 6.8], bromophenol blue). The samples were boiled for 10 min and then cooled to 60°C. Then, 10 μl of proteinase K (Sigma-Aldrich) solution (2.5 mg/ml in lysis buffer) was added and samples were incubated at 60°C for 1 h. Equal volumes of sample and 2× Tricine sample solution (4% SDS, 12% glycerol, 50 mM Tris [pH 6.8], 2% β-mercaptoethanol, 0.01% Serva Blue G [Serva]) were mixed and boiled for 5 min, and then 3 μl per lane was loaded onto a Tricine-buffered polyacrylamide gel (16.5% acrylamide, 6% bis-acrylamide). After electrophoresis, gels were stained by silver staining as described in ref. [56 (link)] and photographed using a Bio-Rad ChemiDoc MP imaging system.
3
Biochemical Reagent Sourcing for Protein Analysis
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Sucrose and mannitol were purchased from BDH Chemicals and Mallickrodt Chemicals, respectively. Bis-Tris, tricine, and amino-caproic acid were purchased from MB Biochemicals (Irvine, CA). Pre-stained SDS-PAGE markers were purchased from Thermo Scientific (Pittsburgh, PA). Bradford protein assay solution and Coomassie brilliant blue (CBB) R-250 were from Bio-Rad laboratories (Richmond, CA). Silver nitrate, streptozotocin (STZ), sodium citrate, NADH, EDTA, n-dodecyl-β-D-maltoside (DDM), and nitro blue tetrazolium (NBT) chloride tablets were obtained from Sigma (St. Louis, MO, USA). Serva Blue G was purchased from Serva (Heidelberg, Germany). Rabbit anti-HNE polyclonal antibodies (IgG) and goat anti-rabbit IgG conjugated with horseradish peroxidase were purchased from US Biological (Salem, MA) and Invitrogen (San Diego, CA), respectively. Hybond-C membrane and a Western blot detection kit were obtained from GE Healthcare (Piscataway, NJ).
4
Isolation and Solubilization of Mitochondrial Membrane Proteins
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Frozen mitochondrial pellets were dissolved in phosphate buffered saline (PBS) supplemented with Complete Mini Protease Inhibitor (Roche Diagnostics Scandinavia AB, Stockholm, Sweden), and the mitochondrial protein concentrations were measured using Nanodrop (Fisher Scientific, Gothenburg, Sweden). Mitochondria were pelleted for 5 min at 5000 g and subsequently dissolved to a concentration of 5 mg protein/ml in MB2 buffer (1.75 M aminocaproic acid, 75 mM Bis-Tris, pH 7.0, 2 mM EDTA, pH 8.0). Mitochondrial membrane proteins were solubilized by incubation with 0.8% digitonin (Sigma Aldrich, Stockholm, Sweden) for 5 min on ice. Samples were centrifuged for 30 min at 13000 g, the supernatant was collected and the protein concentration measured as before. Finally, SBG (750 mM aminocaproic acid, 5% Serva Blue G from SERVA Electrophoresis GmbH, Heidelberg, Germany) was added to a final concentration of 4.5%. These samples were stored at −80°C for electrophoresis.
5
Affinity Purification of Protein Complexes
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Chemicals were from Sigma-Aldrich unless otherwise specified. Detergent n-dodecyl-maltoside (DDM) was from Affimetrix-Anatrace. para-benzophenylalanine was from Bachem. SERVA Blue G and SERVAGel TG PRiME 8–16% precast gels were from Serva. NativePAGE Novex Bis-Tris 3–12% gels were from Life Technologies. Primers are listed in S5 Table and were synthetized by Eurofins and Integrated DNA Technologies. Polyclonal rabbit anti-MBP antibody were from New England Biolabs. Monoclonal mouse anti-RNApol antibody was from BioLegend. Monoclonal M2 anti-FLAG antibody, M2 anti-FLAG agarose beads, and 3xFLAG peptide were from Sigma-Aldrich. CaptureSelect-biotin, Streptavidin DyLight 800, and secondary antibodies goat anti-mouse IgG DyLight 800 conjugate and goat anti-rabbit IgG DyLight 680 conjugate were from Thermo-Fisher.
6
BN-PAGE for Mitochondrial Protein Complexes
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BN-PAGE (Schägger and von Jagow, 1991 (link)) was used for separation of the mitochondrial membrane protein complexes using 6-15% polyacrylamide (w/v) gradient gels with a Mini Protean® 3 System (Bio-Rad Laboratories). Mitoplasts or mitochondria were solubilized in DDM (n-dodecyl β-D-maltoside; Sigma-Aldrich) at a final DDM/protein ratio of 1.0 mg/mg in a buffer containing 1.5 M aminocaproic acid, 2 mM EDTA and 50 mM bis-Tris (pH 7.0) at 4°C. Serva Blue G (Serva) was added to the solubilized protein at a concentration of 0.1 mg/mg detergent and 10 μg protein was loaded into each lane. Electrophoresis was performed at 40 V and 4°C for 1 h and then at 100 V and 4°C.
7
Blue Native PAGE Analysis of Protein Complexes
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Blue native PAGE (BN-PAGE) was carried out as previously described (9 (link)) with some modifications. Crude membranes were resuspended in 1× phosphate-buffered saline (PBS), and protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce). The membranes (10 μg) were solubilized with a final concentration of 1% DMNG for 1 h at 4°C. BN loading dye (5% [wt/vol] Serva Blue G [Serva Electrophoresis GmbH], 250 mM aminocaproic acid, 50% glycerol) (0.5%) was added to the samples, mixed, and run on a NativePAGE Novex Bis-Tris gel system (Thermo Life Technologies), followed by immunoblotting. NC bases were visualized using anti-NC base antibodies and secondary anti-rabbit IgG DyLight 680 conjugate secondary antibodies. FLAG-tagged SpaS was visualized using M2 anti-FLAG antibodies and secondary anti-mouse IgG DyLight 800 conjugate antibodies. Detection was performed using the Odyssey imaging system (Li-Cor).
8
Protein Carbonyl Assay Protocol
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Biotin-linked aldehyde reactive probe ARP) for protein carbonyl assay was from Cayman Chemical (Ann Arbor, MI). Dihydrolipoamide was synthesized from lipoamide in our own laboratory using sodium borohydride as previously described [29] , [30] . ε-amino-N-caproic acid was obtained from MP Biochemicals. Acrylamide/bisacrylamide, ammonium persulfate, Bradford protein assay solution, coomassie brilliant blue (CBB) R-250, immunoblotting membrane, and an ECL immunochemical detection kit were from Bio-Rad laboratories (Richmond, CA, USA). NADH, BSA, lipoamide, EDTA, ATP, and NBT chloride tablets were obtained from Sigma (St. Louis, MO, USA). Serva Blue G was purchased from Serva (Heidelberg, Germany). Anti-PARP antibody was purchased from Trevigen (Gaithers burg, MD). Anti-NQO1 antibodies were from Sigma. Rabbit anti-DLDH polyclonal antibodies (IgG) and goat anti-rabbit IgG conjugated with horseradish peroxidase were purchased from US Biological (Swampscott, MA, USA) and Invitrogen (San Diego, CA, USA), respectively. Other antibodies were from Abcam (Cambridge, MA).
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