Dipeptidyl-peptidase IV (DPP-IV) inhibitory activity was assessed according to the method described by Zeng et al. (12 (link)) with some modifications. Briefly, 25 μL Gly-Pro-p-nitroanilide (6 mM; Sigma-Aldrich) and 25 μL Tartary buckwheat sample, or 25 μL phosphate buffer saline (PBS; Sigma-Aldrich) as a control, were mixed and preincubated at 37 °C for 10 min. The reaction was initiated by adding 50 μL DPP-IV from porcine kidney (3·10–4 U/L, ≥10 U/mg protein; Sigma-Aldrich) and the mixture was incubated at 37 °C for 60 min. The reaction was terminated by adding 100 μL of 1 M sodium acetate buffer (pH=4.0; Sinopharm Chemical Reagent Co., Ltd), and the absorbance of the samples at 405 nm was measured on a Multiskan MK3 plate reader (Thermo Fisher Scientific). Each sample was analyzed in technical triplicate, and the absorbance values were normalized to sample blanks in which DPP--IV was replaced with Tris-HCl buffer (0.1 M, pH=8.0; Solarbio, Beijing, PR China). The negative control (no DPP-IV activity) and positive control (DPP-IV activity with no inhibitor) were prepared by using Tris-HCl buffer (100 mM, pH=8.0; Solarbio) instead of the sample or instead of the DPP-IV solution and the sample, respectively. Diprotin A (Sigma-Aldrich) was used as a standard inhibitor. The DPP-IV inhibition rate was calculated using Eq. 3.
Tris hcl buffer
Manufactured by Solarbio
Sourced in China
Tris-HCl buffer is a commonly used buffer solution in biochemical and molecular biology applications. Its primary function is to maintain a specific pH range, typically around 7.5-8.5, to support the stability and activity of biological molecules and enzymes during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Nonfat milk, by Solarbio (1 mentions)
Anti nr1d1, by Bioss Antibodies (1 mentions)
Anti β actin, by Bioss Antibodies (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Bca kit, by Solarbio (1 mentions)
Traut s reagent, by Sangon (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Ferric chloride hexahydrate, by Merck Group (1 mentions)
Acid phosphatase, by Merck Group (1 mentions)
Superoxide dismutase, by Merck Group (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide edc, by Energy Chemical (1 mentions)
N hydroxysuccinimide, by Energy Chemical (1 mentions)
Fmoc tyr h2po3 oh, by GL Biochem (1 mentions)
Singlet oxygen sensor green (sosg), by Thermo Fisher Scientific (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Sylgard 184, by Dow (1 mentions)
Pyrogallol, by Solarbio (1 mentions)
Potassium bromide, by Solarbio (1 mentions)
13 protocols using tris hcl buffer
1
Dipeptidyl Peptidase-IV Inhibitory Assay
2
Yak Hypothalamus and Testis Protein Expression
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The relative expression patterns of NR1D1 and NR4A2 proteins in HPG from the adult yaks and testis tissues from animals of different ages were examined using Western blot. Total protein was extracted from 100 mg of each tissue sample using RAPI (Solarbio, Beijing, China). Protein concentration was determined using a BCA kit (Solarbio). 100 μg of total protein samples were electrophoresed in a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) for Western blot analysis. The blots were electro-transferred onto a PVDF membrane (Millipore CAT, Billerica, MA, USA), and blocked with Tris-HCl buffer (Solarbio) containing 5 % (w/v) non-fat milk (Solarbio, Beijing, China) for 2 h at room temperature. The membranes were incubated at 4 °C overnight with rabbit monoclonal anti-NR1D1 (1:300), anti-NR4A2 (1:300), and anti-β-actin (1:4000, Bioss, Beijing, China) primary antibodies. The subsequent procedures were carried out as described previously [22 (link)]. All immunoblot assays were performed at least in triplicate. Optical densities of the bands were quantified and scanned using Image-Pro Plus 6.0 (Media Cybernetics Co., Rockville, USA). The expression level of β-Actin was used as an endogenous control. The expression patterns of NR1D1 and NR4A2 proteins in the hypothalamus (4 years old) or testis tissue (2 years old) were used as controls. Data were presented as mean ± SD.
3
Functionalized Nanoparticle Drug Delivery
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CDDP and ALD were purchased from YuanyeBio (Shanghai, China). Cell counting kit 8 (CCK-8), IGEPAL CO-520, hexanol, cyclohexane and Triton X-100 were purchased from Sigma-Aldrich (Missouri, USA) and used as received without purification. Traut’s Reagent was purchased from Sangon Biotech (Shanghai, China). Hydroxyapatite (HAp) was purchased from Macklin (Shanghai, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-Mal) was purchased from Nanocs Incorporation (New York, USA). 1,2-dioleoyl-sn-glycerol-3-phospate (DOPA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[amino(polyethyleneglycol)-2000] (DSPE-PEG-2000), Cholesterol and 1,2-dioleoyl-3-trimethy-lammonium-propane (DOTAP) were purchased and used as received from Avanti (Alabama, USA). Phosphate buffer saline (PBS), ALP activity kit and Tris–HCl buffer were purchased from Solarbio Life Sciences (Beijing, China). Annexin V-FITC (fluorescein isothiocyanate) apoptosis detection kit was obtained from Keygen Biotech (Nanjing, China). Deionized water (DI water) for all experiments was obtained by a purification system (Milli-Q IQ 7000, Massachusetts, USA).
4
Enzyme-Catalyzed Nanoparticle Synthesis
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Ferric chloride hexahydrate (FeCl3·6H2O), ethylene glycol (EG), diethylene glycol (DEG), Acid Phosphatase (AP, MW = 10 kDa, EC 3.1.3.2), Superoxide Dismutase (SOD, ≥ 3000 U mg−1, EC 1.15.1.1) and Chloroperoxidase (CPO, ≥ 3000 U mL−1, EC 1.11.1.10) were purchased from Sigma-Aldrich. Sodium acetate (CH3COONa, NaOAc), Poly (vinylpyrrolidone) (PVP, K30), Pyrogallol, Succinic anhydride and 3-aminopropyltriethoxysilane (APTES) was purchased from Aladdin. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Energy Chemical, Fmoc-Tyr(H2PO3)-OH was purchased from GL Biochem. Tris·HCl buffer was purchased from Beijing Solarbio Science & Technology. Singlet Oxygen Sensor Green (SOSG) was purchased from Thermofisher. The aminophenyl fluorescein (APF) as the HClO detector were purchased from Shanghai Maokangbio. Enhanced BCA Protein Assay Kit was purchased from Beyotime Biotechnology. All materials were used without further purification.
5
Fabrication of Electrochemical Sensors
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Sugar cubes were obtained from Taikoo Sugar Limited (China). PDMS prepolymer and curing agent (Sylgard 184) were purchased from Dow Corning. (3-aminopropyl)triethoxysilane (APTSE) and pyrrole (Py) was purchased from Aladdin. Tris–HCl buffer with a pH of 8.5 were bought from Solarbio. All other chemicals were purchased from Sinopharm Chemical Reagent. All reagents were used without further purification.
6
Aflatoxin detection using aptamer-based assay
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Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxinG1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), zearalenone (ZEA), T-2 toxin, and fumonisin B1 (FB1) were purchased from Qingdao Prebon Biological Engineering Co., Ltd. (Qingdao, China). Soybean oil was purchased from a local supermarket in Changchun, China. AFB1-Apt (5′-FAM-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’) [35 (link)] was synthesized by Shanghai Biotechnology Co., Ltd. (Shanghai, China). Single-walled carbon nanohorns (SWCNHs) were purchased from Xianfeng Nanomaterials Technology Co., Ltd. (Nanjing, China). Tris-HCl buffer (pH 8.8, 1 M) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China), and 1 × TE buffer (pH 7.8–8.2) was purchased from Shanghai Sangong Biotechnology Co., Ltd. (Shanghai, China). A stock solution was prepared by dissolving AFB1-Apt in 0.1 × TE buffer, which was stored in portions at −20 °C. The binding buffer (50 mmol/L Tris-HCl, 10 mmol/L MgCl2, 100 mmol/L NaCl, 5 mmol/L KCl, pH 8.0) was used for a binding reaction between the aptamer and AFB1. All other chemicals were of analytical grade and purchased from commercial suppliers and used without further purification.
7
ELISA Analysis of Perihematoma Tissue
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The ELISA was performed, as previously described [37 (link),38 (link)]. The segment of perihematoma in the ipsilateral cortex was obtained. The dissected tissues were homogenized in ice-cold Tris-HCl buffer (pH = 7.4, Solarbio, Beijing, China) and centrifuged at 13,000 Xg at 4°C for 10 min. ELISA was performed using commercial assay kits shown in Table 1 according to the instructions.
8
Mung Bean Protein Extraction and Characterization
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Peeled mung bean was obtained from Zhanchuang Agricultural Technology Co. Ltd., (Shandong, China). Alcalase (176,743 U/g) was acquired from Novo Co. Ltd., (Beijing, China). 1-anilino-8-naphthalene sulfonate (ANS) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were from Sigma Co., Ltd. (St. Louis, MO USA). Tris-HCl buffer, sodium dodecyl sulphate (SDS), glycerol, bromophenol blue, 2-mercaptoethanol, Coomassie brilliant blue (R-250), trichloroacetic acid (TCA), potassium bromide, pyrogallol, salicylic acid, ferrous sulfate, potassium ferricyanide, ferric chloride, and ferrous chloride were from Beijing Solarbio Technology Co. Ltd. (Beijing, China). All chemicals used in this study were of analytical grade.
9
Synthesis and Characterization of G-Quadruplex DNA
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The oligonucleotides used in this work were synthesized using TaKaRa Clontech to have the sequences
G3, 5′-TGGGAAGGGAGGGAATTCCCT-3′, G4a, 5′-GGGTAGGGCGGGTTGGGAATTCCCA-3′, and
G4b, 5′-GGGTAGGGCGGGTTGGGAATTCCCACCCG-3′.
EcoRI endonuclease (Lot: 10092924), BamHI endonuclease (Lot: 10086715), T4 polynucleotide kinase (Lot: 10088982), endonuclease IV (Lot: 10088353), and Nt.BstNBI (Lot: 10068267) were obtained from New England Biolabs Inc. Hemin (C34H32ClN4O4Fe, Lot: H2028150) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 5-Fluorouracil (Lot: GC22BA0009) was obtained from Sangon Biotechnology Inc. (Shanghai, China). 3,3′,5,5′-Tetramethylbenzidine (C16H20N2, TMB, Lot: SLCF1838) was obtained from Sigma-Aldrich. Tris–HCl buffer (1 M, pH = 7.4, Lot: 20200708) was purchased from Solarbio. Other reagents were of analytical grade and obtained from standard reagent suppliers. All solutions were prepared using Milli-Q water with a resistivity of 18.2 MΩ cm−1. Absorption spectra were recorded using a Purkinje TU-1901 spectrophotometer.
Buffer A used in this work was composed of 50 mM NaCl and 20 mM Tris–HCl buffer, pH 7.4. DNA for the reaction was prepared by combining 10 μL of oligonucleotides (100 μM) with 90 μL of buffer A, annealing the resulting solution at 90 °C for 10 minutes and then cooling it to room temperature, and finally storing the cooled solution at 4 °C.
G3, 5′-TGGGAAGGGAGGGAATTCCCT-3′, G4a, 5′-GGGTAGGGCGGGTTGGGAATTCCCA-3′, and
G4b, 5′-GGGTAGGGCGGGTTGGGAATTCCCACCCG-3′.
EcoRI endonuclease (Lot: 10092924), BamHI endonuclease (Lot: 10086715), T4 polynucleotide kinase (Lot: 10088982), endonuclease IV (Lot: 10088353), and Nt.BstNBI (Lot: 10068267) were obtained from New England Biolabs Inc. Hemin (C34H32ClN4O4Fe, Lot: H2028150) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 5-Fluorouracil (Lot: GC22BA0009) was obtained from Sangon Biotechnology Inc. (Shanghai, China). 3,3′,5,5′-Tetramethylbenzidine (C16H20N2, TMB, Lot: SLCF1838) was obtained from Sigma-Aldrich. Tris–HCl buffer (1 M, pH = 7.4, Lot: 20200708) was purchased from Solarbio. Other reagents were of analytical grade and obtained from standard reagent suppliers. All solutions were prepared using Milli-Q water with a resistivity of 18.2 MΩ cm−1. Absorption spectra were recorded using a Purkinje TU-1901 spectrophotometer.
Buffer A used in this work was composed of 50 mM NaCl and 20 mM Tris–HCl buffer, pH 7.4. DNA for the reaction was prepared by combining 10 μL of oligonucleotides (100 μM) with 90 μL of buffer A, annealing the resulting solution at 90 °C for 10 minutes and then cooling it to room temperature, and finally storing the cooled solution at 4 °C.
10
Hepatic Biomarker Evaluation in Rats
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The hepatic tissues of rats were homogenized in Tris–HCl buffer (pH 7.4, Solarbio, Beijing, China). Using a Sigma 1–14 K centrifuge (Sigma, Germany), blood samples were collected from the abdominal aorta for a nest assay and subsequently centrifuged at 3,000 rpm for 15 min to obtain serum samples. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), triglycerides (TGs), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), fatty acid synthetase (FAS), lipase (LIPA) and high-density lipoprotein cholesterol (HDL-C) were measured using commercial detection kits and a microplate reader (Thermo, USA). Serum TC, TG, and LDL-C kits were purchased from Zhicheng Biological Technology Co., Ltd. (Shanghai, China). Liver TC, TG, and LDL-C kits and FAS and LIPA kits were obtained from Shanghai Sino Best Biological Technology Co., Ltd. (Shanghai, China). HDL-C, SOD, MDA, GSH-Px, GSH, AST, and ALT kits were obtained from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China).
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