Affinity-purified DP S-tag was retained on agarose beads and washed three times with 100 µl of water. DP S-tag was first cleaved C-terminal to Met using cyanogen bromide (Sigma-Aldrich). The DP S-tag beads were suspended in 50 µl of 70% TFA (Sigma-Aldrich) in water and one small crystal (<1 mg) of cyanogen bromide was added to the solution. The reaction proceeded 20 h at 4°C. After cleavage, the supernatant containing peptides was collected and dried down in a centrivap concentrator (Labconco). The sample was then resuspended in 30 µl of 100 mM ammonium bicarbonate (Sigma-Aldrich) and AspN (Roche) was added at an enzyme/substrate ratio of 1:20. The digestion was performed for 6 h at room temperature and then quenched by adding acetic acid (Sigma-Aldrich) to adjust the pH to 3.
Cyanogen bromide
Manufactured by Merck Group
Sourced in United States
Cyanogen bromide is a chemical compound commonly used in various laboratory applications. It serves as a reagent for the cleavage of peptide bonds in proteins during protein analysis and purification procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium bicarbonate, by Merck Group (2 mentions)
Sodium azide, by Merck Group (2 mentions)
Trifluoroacetic acid tfa, by Merck Group (2 mentions)
1 1 bi 2 naphthol, by Merck Group (1 mentions)
Bisphenol a, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Abcam (1 mentions)
N n diisopropylethylamine, by Merck Group (1 mentions)
Reverse phase high performance liquid chromatography rp hplc, by Waters Corporation (1 mentions)
Ethanol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
N methyl 2 pyrrolidone, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
β mercaptoethanol β me, by Merck Group (1 mentions)
1 anilino 8 naphthalene sulfonic acid, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Sodium tetrathionate, by Merck Group (1 mentions)
10 protocols using cyanogen bromide
1
Protein Purification and Cleavage Protocol
2
Synthesis of Organic Compounds
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Bisphenol A (BPA), 1,1-bi-2-naphthol (BN),
phenol, formaldehyde, oxalic acid catalyst, cyanogen bromide (BrCN),
and iodine were purchased from Sigma-Aldrich Chemical and were used
directly. Tetrahydrofuran (THF) was refluxed over sodium and benzophenone
under a nitrogen atmosphere for more than 2 days before distillation
use. Triethylamine was distilled from CaH2 under a nitrogen
atmosphere before use.
phenol, formaldehyde, oxalic acid catalyst, cyanogen bromide (BrCN),
and iodine were purchased from Sigma-Aldrich Chemical and were used
directly. Tetrahydrofuran (THF) was refluxed over sodium and benzophenone
under a nitrogen atmosphere for more than 2 days before distillation
use. Triethylamine was distilled from CaH2 under a nitrogen
atmosphere before use.
3
Purification and Kinase Assay Protocol
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Purified Etk, Btk, Itk, Mer, Yes, Lyn and Src kinases were obtained from Millipore Inc (Dundee, UK). PI, N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), ethanol, acetonitrile (ACN), N-Methyl-2-pyrrolidone (NMP), 1-(3-aminopropyl)piperidine, trifluoroacetic acid (TFA), Pd/C, ammonium formate, cyanogen bromide and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). L -Leucine methyl ester hydrochloride and 4-(4-pyridinyl)benzoic acid were purchased from Chem-Impex International Inc (Wood Dale, IL, USA). The Annexin V-FITC apoptosis detection kit was obtained from Abcam (Cambridge, MA, USA). Reverse-phase high-performance liquid chromatography (RP-HPLC) from the Waters Corporation (Milford, MA, USA) was used for analysis and purification of CTN06.
4
Purification and Characterization of Insulin
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1-anilino-8-naphthalene sulfonic acid (ANS), cyanogen bromide (CNBr), kanamycin, isopropyl β-D-1 thiogalactopyranoside (IPTG), β-mercaptoethanol (β-ME), and other chemicals were purchased from Sigma. The dialysis tubes with molecular weight cut-off (MWCO) of 2 and 12 kDa were purchased from Spectrum Scientific Company. The gel filtration media were purchased from GE Healthcare and Qiagen. Insulin (INS) ELISA kit was purchased from Shanghai Crystal day Biotech Co., Ltd.
5
Protein Aggregation Assay Protocol
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Bis-1-anilino-8-naphthalene sulfonate (bis-ANS), thioflavin T (ThT), sodium tetrathionate, sodium sulfide, cyanogen bromide, and other chemicals were purchased from Sigma. Standard insulin and goat anti-rabbit IgG peroxidase were purchased from Sigma-Aldrich Company. The dialysis tube (2 kDa cut-off) was purchased from Spectrum Scientific Company. Anti αB-Cry antibody was a generous gift of Professor Samuel Zigler (Johns Hopkins School of Medicine). Gel filtration media and Ni-NTA matrix were from GE Healthcare and Qiagen, respectively.
6
Solid-Phase Peptide Synthesis and Caspase Assay
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All solvents and reagents were used as supplied. Fmoc amino acid derivatives were purchased from Novabiochem (Billerica, MA, USA). 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluro noium hexafluorphosphate (HATU), formic acid, trifluoroacetic acid (TFA) and N,N’-diisopropylcarbodiimide (DIC) were obtained from IrisBiotech. TentaGel® HL-NH2 resin (0.56 mmol/g, 110 μm particle size) was purchased from RappPolymere (Tuebingen, Germany). Cyanogen bromide (3M solution in dichloromethane), 1,4-diazabicyclo[2.2.2]octane (DABCO), iodoacetic acid, triisopropylsilane (TIS), Pipes (2,2′-(Piperazine-1,4-diyl)di(ethane-1-sulfonic acid)), ethylenediaminetetraacetic acid (EDTA), DL-dithiothreitol (DTT), caspases 3 and 7 and solvents for peptide synthesis (N,N-dimethylformamide (DMF), dichloromethane (DCM), and (N-ethyldiisopropylamine (DIEA)) were obtained from Sigma Aldrich (St. Louis, MO, USA).
7
Synthesis and Characterization of Dicyanate Ester Monomer
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The dicyanate ester monomer 2,2-bis(4-cyanatophenyl)propane (1) (Fig. 1 ) was supplied by Lonza AG (Visp, Switzerland) (as PRIMASET™ BADCy) and, having confirmed its purity using 1H NMR and elemental analysis, was used as received without further purification. In the interests of brevity, the analytical data (from FTIR ATR, 1H and 13C NMR spectroscopy) are given as ESI.† The synthesis of l,l-dichloro-2,2-(4-cyanatophenyl)ethylidene was based on a well-used route, originally reported by Grigat and Pütter17 (link) Copper(ii ) acetylacetonate, Cu(acac)2, (97%) and dodecylphenol (mixture of isomers) (used in the co-catalyst package) and cyanogen bromide (97%) were obtained from Sigma Aldrich. Bisphenol C (l,l-dichloro-2,2-(4-hydroxyphenyl)ethylidene, 97%) was obtained from AOKChem, Shanghai. All reagents were analysed to determine purity and used as received without further purification.
8
Rabbit Immunization for CB Antibodies
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Three rabbits from the New Zealand strain were immunized over three months in intervals of fifteen days, with increasing doses of CB (10–300 μg/rabbit) alternating with incomplete Freund's adjuvant and alum (aluminum hydroxide and magnesium hydroxide, Thermo Fisher). The serum of the three rabbits was mixed and the antibodies were purified by affinity chromatography in a Sepharose 4B resin activated with cyanogen bromide (Sigma) and coupled to CB (5 mg).
9
Functionalization of Biopolymers for Biosensing
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(3-Glycidyloxypropyl) trimethoxysilane (GPTS,98%), triethyl amine (99%), cyanogen bromide (CNB, 97%), divinyl sulfone (DVS; 97%), carbonyl diimidazole (CDI, 90%), hydrochloric acid (37%), hydrogen peroxide (30%), sodium azide, citric acid, sulfuric acid (96%), bovine serum albumin (BSA), horseradish peroxidase (HRP), ochratoxin A (OTA) and sephadex ™ G25 were all purchased from Sigma Aldrich (France) and used without further purification. Phosphate buffered saline (PBS 10×) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) chromogen/substrate solution for ELISA were purchased from Invitrogen. The peptides were synthesized by Smartox (France).
10
Porcine Pancreatic Elastase Purification
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Porcine pancreatic elastase (PE) as well as DES and IDES standards were obtained from Elastin Products Company (Owensville, MO, USA). Propionic anhydride solution, ammonium bicarbonate, cyanogen bromide, 2-mercaptoethanol, urea, sodium azide, sodium chloride, and trypsin from porcine pancreas were obtained from Sigma Aldrich (Steinheim, Germany). Hydrochloric acid was purchased from Grüssing GmbH (Filsum, Germany) and analytical grade Tris, trifluoroacetic acid (TFA), and formic acid (FA) were obtained from Merck (Darmstadt, Germany). HPLC-grade acetonitrile (VWR Prolabo, Leuven, Belgium), HPLC-grade methanol (VWR Prolabo), and doubly distilled water were used. All other chemicals were of analytical grade or higher purity.
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