Sodium phosphate, sodium chloride, triazine dye Cibacron Blue 3GA and the protease substrates N-benzoyl-L-tyrosine ethyl ester (BTEE) and Nα-benzoyl-L-arginine ethyl ester (BAEE) were obtained from Sigma-Aldrich (Darmstadt, Germany). Potassium thiocyanate (KSCN) was obtained from Carlo Erba (Chaussée du Vexin, France). Hydrogen chloride (HCl) was obtained from Scharlab (Barcelona, Spain). All other chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany). The structures were created using ChemDraw Ultra 12.0 (CambridgeSoft, Cambridge, MA, USA, www.cambridgesoft.com ).
Cibacron blue 3ga
Manufactured by Merck Group
Sourced in United States, Germany
Cibacron Blue 3GA is a laboratory reagent used in affinity chromatography. It is a synthetic dye molecule that can selectively bind to certain proteins, enzymes, and other biomolecules, allowing their isolation and purification from complex mixtures. The core function of Cibacron Blue 3GA is to provide a versatile and effective tool for the separation and purification of a wide range of biomolecules in research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
N benzoyl l tyrosine ethyl ester, by Merck Group (1 mentions)
Nα benzoyl l arginine ethyl ester, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Reduced glutathione, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Hydroxylamine, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Cumene hydroperoxide, by Merck Group (1 mentions)
Manganese 2 chloride, by Merck Group (1 mentions)
Deae cellulose, by Merck Group (1 mentions)
Bacto tryptone, by Merck Group (1 mentions)
Chromatography resins, by GE Healthcare (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dna ligase, by New England Biolabs (1 mentions)
9 protocols using cibacron blue 3ga
1
Spectrophotometric Enzyme Kinetics Protocol
2
Taro Corm Extract Purification Protocol
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Colocasia esculenta (L.) Schott corms were manually chosen and purchased from a local market in Rio de Janeiro, Brazil. The crude taro extract (CTE) was obtained according to Roy, Banerjee, Majumder, & Das [14 (link)] and was stored at –20°C until tarin purification steps. Tarin purification was performed according to the protocol described previously by Pereira et al. [7 (link)], by affinity chromatography through a Cibacron Blue 3GA (Sigma-Aldrich Co, MO, USA) column. Protein concentrations of the tarin fractions were estimated by the Lowry method [15 (link)], using bovine serum albumin (BSA) (Sigma-Aldrich Co) at 1mg/mL for the standard curve.
3
Oxidative Stress Response Assays
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NADPH, H2O2, ter-butyl hydroperoxide solution (Luperox), cumene hydroperoxide, Trizol®, bacto yeast, bacto tryptone, IPTG, ampicillin, and chloramphenicol, as well as Tris, EDTA, oxidized glutathione (GSSG), reduced glutathione (GSH), PMSF, manganese (II) chloride, L-glutamine, hydroxylamine, ADP, DEAE-cellulose, HA-Ultrogel, and Cibacron Blue 3G-A were obtained from Sigma-Aldrich, Merck KGaA, (Darmstadt, Germany). All other chemicals were purchased from JT Baker Chemical, Phillipsburg, NJ, USA.
4
Cibacron Blue Affinity Resin Synthesis
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Chromatography resins were obtained from GE Healthcare. The Cibacron-blue affinity resin used for SecA purification was made by alkaline coupling of Cibacron Blue 3GA (Sigma) to Sepharose CL-6B. Approximately 145 g of hydrated Sepharose CL6B was mixed with 220 mL of 0.5 M NaCl and 8.4 g of Cibacron Blue dye; 9 mL of 10 M NaOH was added and the suspended resin was mixed on a shaker table for 60 minutes at 37 °C. The resulting blue gel was washed successively with 1 M NaOH, H2O, 60% ethanol, H2O, and finally 20% ethanol for storage.
5
Purification of Human Transthyretin
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Human plasma was pretreated by reduction of albumin via adsorption in a Cibacron blue 3GA (Sigma-Aldrich, St. Louis, MO, USA) column. The unbound faction was concentrated by ultrafiltration. Human TTR was purified from the concentrated, pretreated human plasma by preparative discontinuous native-PAGE using BIO-RAD Model 491 Prep Cell system (BIO-RAD, Hercules, CA, USA) as described previously [17 (link)].
6
Taro Extract Purification and Characterization
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Colocasia esculenta corms were purchased at a local market in the Niterói municipality (22°52′51″ S, 43°6′15″ W), Southeastern Brazil. The crude taro extract was obtained according to Roy et al. [41 (link)], with modifications, and fractionated using the affinity chromatography resin Cibacron Blue 3G-A (Sigma-Aldrich Co, St. Louis, MO, USA), as described by Pereira et al. [10 (link)]. In order to reduce interferences in subsequent in vitro and in vivo experiments, the Tris-HCl present in the purified fraction containing tarin was removed by dialysis (Fisherbrand, Pittsburgh, PA, USA) against water at 8 °C for 18 h under constant stirring and lyophilized (Labconco, Kansas, MO, USA) for storage. Protein quantification in the crude extract was performed by the Peterson [23 (link)] method, using bovine serum albumin (BSA) as standard.
7
Molecular Biology Reagent Specifications
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Oligonucleotide primers were synthesized by Operon (Eurofins MWG Operon, Huntsville, AL, USA). PCR and plasmid purification kits were from GibcoBRL (Long Island, NY, USA) and Qiagen (Hilden, Germany), and DNA ligase was from New England Biolabs (Ipswich, MA, USA). Taq DNA polymerase was a product of Invitrogen (Carlsbad, CA, USA). Horseradish peroxidase conjugated antirabbit IgG and the ECL kit were from Calbiochem (San Diego, CA, USA) and Amersham Bioscience (Chalfont St Giles, UK), respectively. Human RBP purified from urine was from RDI (Fitzgerald, MA, USA). Cibacron blue 3GA were products of Sigma (St Louis, MO, USA). Human Aβ 1-142 was from Millipore (KGaA, Darmstadt, Germany), Alexa Fluor 488 TFP ester from Molecular Probes (Life technologies, USA), and Bio-gel P2 from Bio-Rad (CA, USA). All of other chemicals used were of the analytical grade.
8
Protein deglycosylation protocol
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Polyethylene glycol Mw=1000 (PEG1000) (Lot A0319044), Polyethylene glycol (Lot 1356267) Mw=8000 (PEG8000) (Lot SLBC9317V), Ammonium sulfate (NH4)2SO4, dipotassium hydrogen orthophosphate K2HPO4, potassium di-hydrogen orthophosphate KH2PO4, Tris (hydroxymethyl)-aminomethane (Tris) were purchased from Fisher Scientific UK (Leicester, UK). Dextran500 from Leuconostoc mesenteroides (Lot BCBJ7122V) weightaverage molecular weight (Mw) 450,000-650,000 and Ovalbumin from lyophilized powder, >98% (Lot SLBD2312V), Endoglycosidase F1 from Elizabethkingia miricola; recombinant, expressed in E.coli, ≥16U/mg buffered aqueous solution (Lot SLBK8022V), Neuraminidase from Clostridium perfringens (C. welchii) type V from lyophilized powder 0.32 mg solid 7.9U/mg (N2876-2.5UN Lot SLBF5907V), Alkaline phosphatase (ALP) from bovine intestinal mucosa-BioUltra, in buffered aqueous glycerol solution 2000-4000 DEA U/mg protein (Lot SLBF3716V) and Cibacron Blue 3G-A (product No.C9534-25G) were purchased from Sigma-Aldrich UK (Dorset, UK).
9
Purification and Characterization of Phthalate Degrading Enzymes
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All steps were carried out under anoxic conditions (95% N 2 , 5% H 2 ). Around 3 g of phthalate grown cells (wet mass) were suspended in 9-12 ml buffer A and lysed using a French pressure cell. After ultracentrifugation, the supernatant was filtered through a 0.2-μm sterile filter (Filtropur S 0.2, Sarstedt) and applied to a 12 ml DEAE-Sepharose column (GE Healthcare), equilibrated with buffer A at a flow rate of 0.5 ml min -1 . The column was washed with two bed volumes of buffer A and with buffer A supplemented with 15 mM KCl (PCD) or 20 mM KCl (PCL). Fractions eluting in buffer A at higher KCl concentrations as indicated in the results section were tested for PCL/PCD activities. Activity containing fractions were concentrated and used for SDS-PAGE analysis and enzymatic assays. Other chromatography materials tested for PCL/PCD enrichment were Resource Q-Sepharose anionic exchanger, Superdex 200 10/300 Gl gel filtration (all GE Healthcare), and the affinity dyes Reactive Green 5 Agarose, Reactive Red 120, and Cibacron Blue 3GA (1 ml columns each, all Sigma-Aldrich). All columns were equilibrated with buffer A, and elution was by varying KCl, or in case of the affinity dyes, phthalate concentrations (0.1-1 M).
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