Total protein extracts were prepared from tail biopsies minced in 150 μL Tris–HCl, pH 7.8, and subjected to sonication and freeze-thawing cycles. For protein extracts from total bone, the tissue was frozen in liquid nitrogen and homogenized using aluminum beads and the TissueLyser II instrument (Qiagen, Hilden, Germany). Tissue debris were removed by centrifugation at 4°C. Protein concentration in the supernatants were estimated using the DC™ Protein Assay Kit II (Bio-Rad, Berkeley, CA, USA) following the manufacturer’s instructions, on a Synergy™ H4 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). DPP3 enzymatic activity was performed as reported,(31 (link)) with minor modifications. Briefly, 50 to 80 μg of protein extracts were assayed with 0.04 mM Arg-Arg-β-naphthylamide (Sigma-Aldrich, Saint Louis, MO, USA) in 250 μL Tris–HCl, pH 8.5, at room temperature (RT). The reaction was stopped by adding 50 μL of 2 M acetate buffer, pH 4.5, containing 10% Tween and 1.5 mg/mL Fast Blue B Salt (all chemicals from Sigma-Aldrich). The absorbance of the released product (β-naphthylamine) was measured at 530 nm, using SynergyTM H4 Microplate Reader. The enzymatic activity was expressed as percentage of the wild-type (WT) value.
Fast blue b salt
Manufactured by Merck Group
Sourced in United States, Germany, Poland
Fast blue B salt is a chemical compound used as a reagent in various laboratory applications. It is a diazonium salt that can be used for the detection and visualization of certain organic compounds. The core function of Fast blue B salt is to act as a coupling agent in histochemical and biochemical assays.
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Lab products found in correlation
α naphthyl acetate, by Merck Group (7 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (6 mentions)
L glutathione reduced, by Merck Group (5 mentions)
1 naphthyl acetate, by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (4 mentions)
Gallic acid, by Merck Group (3 mentions)
β naphthol, by Merck Group (3 mentions)
α naphthol, by Merck Group (3 mentions)
Eserine, by Merck Group (3 mentions)
Acetylthiocholine iodide, by Merck Group (3 mentions)
Arg arg β naphthylamide, by Merck Group (2 mentions)
Proteinase k from tritirachium album limber, by Roche (2 mentions)
Subtilisin a from bacillus licheniformis, by Merck Group (2 mentions)
Fluorescein isothiocyanate fitc labeled casein, by Thermo Fisher Scientific (2 mentions)
Ethyl acetate, by Merck Group (2 mentions)
Coomassie brilliant blue g 250, by Merck Group (2 mentions)
α naphthyl acetate α na, by Merck Group (2 mentions)
β naphthyl acetate, by Merck Group (2 mentions)
Folin ciocalteu reagent, by Merck Group (2 mentions)
30 protocols using fast blue b salt
1
Protein Extraction and DPP3 Activity Assay
2
Osteogenic Differentiation of ADMPCs
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To induce osteogenic differentiation, ADMPC were cultured in 48 well plate until confluent and were then treated with osteogenic differentiation medium (ODM) consisting of MF medium supplemented with, 10 mM b-glycerophosphate, 50 µg/ml ascorbic acid, 10 nM dexamethasone, and 200 ng/ml recombinant human bone morphogenetic protein (rhBMP)-2 (R&D systems), as described previously45 (link). ADMPC without ODM served as a control group. Extracellular matrix calcification was determined on fixed ADMPC stained with 1% alizarin red solution (Wako) for five mins. ALP activity was detected by staining with ALP staining solution consisting of 0.1 mg/ml naphthol AS-MX phosphate (Sigma Aldrich), 0.6 mg/ml Fast Blue B salt (Sigma Aldrich), 2 mmol/L MgCl2 (Wako), 0.5% N,N dimethylformamide (Wako) and Tris-HCl (pH 8.5) at room temperature as described previously46 (link). Photographs were obtained to compare the intensity of staining between the control and ODM cultures.
3
Enzyme and Organism Procurement Protocol
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TransStart® TopTaq DNA Polymerase was purchased from TransGen Biotech (Beijing, China). Nde I, Not I, BamH I and Bgl II endonuclease were purchased from Takara (Dalian, China). Proteinase K from Tritirachium album limber were purchased from Roche (Mannheim, Germany). Subtilisin A from Bacillus licheniformis, N-acetyl-D,L-phenylalanine-β-naphthylester and Fast Blue B Salt were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-labeled casein was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Escherichia coli BL21(DE3) competent cells were purchased from Sangon Biotech (Shanghai, China). E. coli Origami 2(DE3), B. bassiana, Saccharomyces cerevisiae and Candida albicans were all preserved by the College of Biological Science and Engineering, Shaanxi University of Technology.
4
Visualizing Protease Inhibitor Activity
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The activities of protease inhibitors were visualized by the method of Uriel and Berges [23 (link)], with a slight modification. Equal amounts (5 μg) of proteins (extracted according to section 2.2) were separated by the native polyacrylamide gel electrophoresis (PAGE). After electrophoresis, the gels were incubated at 37°C for 20 min with proteinase K solutions (0.07 mg/mL, Sigma–Aldrich, USA) in 0.1 M phosphate buffer, pH 7.4. Specific inhibitory activity toward proteinase K was visualized by staining the gels with the Fast Blue B Salt (Sigma–Aldrich) solution containing N-acetyl-dl-phenylalanine-b-naphthyl ester (Sigma–Aldrich).
5
Phytochemical Analysis and Bioactivity Evaluation
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All solvents and chemicals used were of analytical reagent grade. 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH•) free radical, iron (III) chloride (97%), gallic acid (97%), β-sitosterol (95%), and Gram’s iodine solution were purchased from Sigma-Aldrich (Munich, Germany). α-Amylase from Bacillus licheniformis liquid (Cat. No. A4862), AChE from Electrophorus electricus, Fast Blue B Salt, and soluble starch were obtained from Sigma-Aldrich (Munich, Germany). Ethyl acetate, ethanol, ferric chloride (97%), methanol, sodium dihydrogen phosphate (NaH2PO4) and disodium phosphate (Na2HPO4) were obtained from Merck (Darmstadt, Germany). Acarbose was from Bayer (Leverkusen, Germany), n-hexane from BDH (Poole, England), p-anisaldehyde from ACROS organics (New Jersey, USA), and donepezil from Cayman chemicals (Ann Arbor, MI, USA). Bovine serum albumin (BSA) was purchased from PAA Laboratories GmH (Haidmannweg, Austria). Tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl) buffer solution was obtained from Calbiochem (San Diego, CA, USA), and Milli-Q water (Millipore®, Merck, Darmstadt, Germany) was used to prepare all solutions. All chromatographic separations were performed on 20 × 10 cm normal phase Silica gel 60 F254 HPTLC glass plates (Merck, Darmstadt, Germany).
6
Serum Dpp3 Enzymatic Activity Assay
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Dpp3 enzymatic activity in the sera of patients and controls was measured as previously reported [7 (link)], with minor modifications. Briefly, serum protein concentration was determined using the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Then, a volume of serum corresponding to about 50 μg of total proteins was assayed with 0.04 mM Arg-Arg-β-naphthylamide (Sigma-Aldrich, St. Louis, MO, USA) in Tris-HCl, pH 8.5, at 37 °C. The reaction was stopped by adding 2 M acetate buffer, pH 4.5, containing 10% Tween and 1.5 mg/mL Fast Blue B Salt (all chemicals from Sigma-Aldrich). The absorbance of the released product (β-naphthylamine) was measured at 530 nm, using a SynergyTM H4 Microplate Reader. The enzymatic activity was expressed as nmol of β-naphthylamine (2-NA)/mg proteins/min).
7
Isothiocyanate Compounds and Enzyme Assays
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Allyl isothiocyanate (AITC), 2-phenylethyl isothiocyanate (2PEITC), 3-butenyl isothiocyanate (3BITC) and 3-(methylthio) propyl isothiocyanate (3MPITC) (Figure 2 ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The purities of AITC, 2PEITC,3BITC and 3MPITC were 99.7, 99, 97 and 98%, respectively. The following chemicals were purchased from Sigma-Aldrich as well for enzyme activity assay: Coomassie Brilliant Blue G-250, α-naphthol, β –naphthol, α-naphthyl acetate (α-NA), β-naphthyl acetate (β-NA), β-nicotinamide adenine dinucleotide phosphate (β-NADPH), fast blue B salt, 1-chloro-2,4-dinitrobenzene (CDNB), l -glutathione reduced (GSH), acetylthiocholine iodide (ATC), 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB).
8
Antioxidant and Cholinesterase Inhibition Assays
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Fast Blue B salt, 2,2- diphenyl-1-picrylhydrazyl radical (DPPH, 97%), butyrylcholinesterase (BChE, from equine serum, ≥140 U/mg) and acetylcholinesterase (AChE, from Electrophorus electricus Linnæus, ≥245 U/mg), 1-naphthyl acetate, gallic acid, Trolox and solvents/reagents of analytical grade were purchased from Fluka Sigma-Aldrich, Schnelldorf, Germany. Bioluminescent marine Aliivibrio fischeri bacteria (NRRL-B11177, DSM no. 5171) were obtained from the German Collection of Microorganisms and Cell Cultures, Berlin, Germany. Ethanol, methanol and MS grade solvents (Optima LC-MS grade) methanol and acetonitrile were provided from Thermo Fisher Scientific, Schwerte, Germany. Bacillus subtilis spores (BGA, DSM 618 strain), formic acid (96%), hydrochloric acid, ethyl acetate, butanone, Folin-Ciocalteu reagent and HPTLC plates silica gel 60 F254 were obtained from Merck, Darmstadt, Germany. Thiazol blue tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, MTT) were purchased from Carl Roth, Karlsruhe, Germany. Bidistilled water was prepared with a Destamat Bi 18E, Heraeus, Hanau, Germany. formic acid (98%) was from J.T. Baker, Deventer, Netherlands.
9
Insecticide Resistance Enzyme Assays
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Imidacloprid (95.6%) was purchased from Dupont. Beta-cypermethrin (95%) was obtained from Suzhou Fumeishi Chemical Co., Ltd. Chlorpyrifos (98%) was obtained from Tianjin Longdeng Chemical Co., Ltd. Chlorfenapyr (98%) was obtained from Jiangsu Academy of Agricultural Sciences. Acetamiprid (90%) was provided by Jiangsu Yangnong Chemical Group Co., Ltd. Azamethiphos (95%) was supplied by Shanghai Yongyuan Chemical Co., Ltd. Piperonyl butoxide (PBO, 90%), s,s,s-tributylphosphorotrithioate (DEF, 98%) and diethyl maleate (DEM, 97%) were purchased from Chem. Service (West Chester, PA). α-Naphthyl acetate (α-NA), β-naphthyl acetate (β-NA), eserine, α-naphthol, β-naphthol, 1-chloro-2,4-dinitrobezene (CDNB), reduced glutathione (GSH), phenylmethylsulfonyl (PMSF), dithiothreitol (DTT), phenylthiourea (PTU), fast blue B salt, sodium dodecyl sulfate (SDS) and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO) at the highest purity available. The other chemicals were of analytical quality and purchased from commercial suppliers.
10
Enzyme Expression in E. coli Strains
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E. coli Trans-T1, BL21(DE3), and Origami 2(DE3) were purchased from TransGen Biotech (Beijing, China), Sangon Biotech (Shanghai, China) and Invitrogen (Carlsbad, CA, USA), respectively. TransStart® FastPfu DNA polymerase (FastPfu), TransStart® FastPfu Fly DNA polymerase (FastPfu Fly) and EasyPfu DNA Polymerase (EasyPfu) were purchased from TransGen Biotech. Proteinase K from Tritirachium album limber and elastase from the porcine pancreas were purchased from Roche (Mannheim, Germany) and Sangon Biotech, respectively. Subtilisin A from Bacillus licheniformis, trypsin and α-chymotrypsin from the bovine pancreas, N-acetyl-D,L-phenylalanine-β-naphthylester (APNE) and Fast Blue B Salt were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-labeled casein was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant expression plasmids BmSPI38-p28 and BmSPI39-p28 were preserved by the College of Biological Science and Engineering, Shaanxi University of Technology.
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