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Human angiotensin 2

Manufactured by Merck Group
Sourced in United States

Human angiotensin II is a laboratory reagent used for research purposes. It is a peptide hormone that plays a role in the regulation of blood pressure and fluid balance in the human body. The product is intended for use in scientific research and experimentation, and its core function is to serve as a tool for studying the physiological effects of angiotensin II.

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21 protocols using human angiotensin 2

1

Transient Transfection and Luciferase Assay

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HEK 293 T cells were seeded into T75 flasks at 90,000 cells/cm2 and incubated at 37 °C and 5% CO2 overnight. The following day, cells were transiently transfected for 48 hours in total; for the last 24 hours, cells were plated at 10,000 cells/well into sterile, white, 384-well plates (Nunc/Thermo Fisher Scientific) containing 5–10 mg/ml of EMR2 antibody or 25 μM AngII (human Angiotensin II, Sigma-Aldrich) in phenol-red free OptiMEM1 medium (Gibco/Thermo Fisher). Luminescence was measured using the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA) as per manufacturer’s instructions. The Luminescent signal was read on a EnVision imager (Perkin-Elmer).
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2

Eucommia Lignans Proliferation and Apoptosis

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Human angiotensin II was purchased from Sigma-Aldrich (St. Louis, MO, USA). The lignan content in Eucommia lignans was determined to be 71% by spectrophotometry on a Beckman Coulter DU 640 spectrophotometer (Beckman Coulter, Inc., USA) at 277 nm; the lignans were extracted from Eucommia ulmoides Oliv. bark in our own laboratory using a previously described method [2 (link)].
The primers for P21, P27, Bax, Bcl-2, and AR were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against P21, P27, Bax, Bcl-2, and AR for western blotting were supplied by Abcam (Cambridge, England) and Santa Cruz (CA, USA). The Cell Titer 96 Aqueous One Solution Proliferation Assay for the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method was purchased from Promega (Madison, WI, USA). The RT-qPCR kits with Platinum SYBR Green qPCR Super Mix-UDG and flow cytometry were purchased from Invitrogen (Carlsbad, CA, USA). Epalrestat was obtained commercially from Yuancheng Pharmaceutical Co., Ltd. (Hubei, China). The MTT One-Step Cell Proliferation Test Kit and Annexin V-FITC Apoptosis Assay Kit were purchased from the Promega Corporation and Bibo Biological Technology Co., Ltd. (Nanjing, China), respectively.
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3

Synthesis and Characterization of CPPM Compound

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Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific and DMSO-d6 was purchased from Cambridge Isotopes Laboratory, Inc. PBS Buffer, 10x, pH 7.4, was purchased from Thermo Fisher Scientific, Inc. and diluted to 1x concentration with Milli-Q water. Trifluoroacetic acid (TFA) was purchased from Alfa Aesar and used without further purification. Methanol and dichloromethane were purchased from Fisher Scientific and used without further purification. Additional materials required for the synthesis of the CPPM used in this study, MePh10-b-dG5, have been previously reported.43 (link),44 (link) Human angiotensin II (#A9525), bradykinin (#B3259), diethylpyrocarbonate (DEPC) (#D5758), imidazole (#I5513), sodium chloride (#S5886), guanidine thiocyanate (GTC, #G6639), and iodoacetamide (#I6125) were all purchased from Sigma-Aldrich (St. Louis, MO). Immobilized TPCK-trypsin (#20230) was obtained from Thermo Scientific (Waltham, MA). Potassium phosphate monobasic (#P285), sodium phosphate dibasic anhydrous (#S374), formic acid (#A117), water (#W7), and acetonitrile (#A998) were obtained from Fisher Scientific (Fair Lawn, NJ). Amicon® centrifugal filters (#UFC501096 & #UFC800324) were from Millipore Sigma (Burlington, MA). Superfolder green fluorescent protein (sfGFP) was expressed and purified by Dr. Francesca Anson using procedure described below.
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4

Peptide Characterization by Mass Spectrometry

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Water, methanol, and trifluoroacetic acid (TFA) used as solvents and additives throughout these experiments were obtained from Fisher Scientific (Optima LC/MS grade). The modified peptide renin substrate I (97%), human angiotensin I (90%), human angiotensin II (93%), bradykinin (98%), substance P (95%), Met5-enkephalin (95%), and [d-Ala2,d-Leu5]-enkephalin (95%) were all purchased as acetate salts from Sigma-Aldrich (St. Louis, MO). Biotinylated, phosphorylated SAMS peptide was obtained from Enzo Life Sciences (Farmingdale, NY). Support solutions for the SCGD were prepared by dilution of concentrated, trace metal grade hydrochloric acid (J.T. Baker, Center Valley, PA) with deionized water of 18.2-MΩ resistivity, prepared in-house with a mixed-bed ion-exchange deionization unit.
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5

Irbesartan and Angiotensin II Preparation

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Irbesartan (Sigma-Aldrich Corp, St. Louis, MO) and human angiotensin II (Sigma-Aldrich Corp) were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Corp) and prepared as a concentrated stock. DMSO was used as a vehicle for the purpose of concentrated stock preparation for experiments without any effects on HTFs. For this study, the drugs were diluted further with culture medium to achieve the concentrations desired by the experimental design. The final concentrations of DMSO in experimental solutions were 0.04% for 10 μg/mL Irbesartan, 0.2% for 50 μg/mL Irbesartan, 0.4% for 100 μg/mL Irbesartan, and 0.2% for 2 μg/mL angiotensin.
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6

Angiotensin II and Bradykinin Quantification

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C57BL/6 J male mice aged 8 weeks were purchased from Japan SLC (Hamamatsu, Japan). All animal experiments were approved by the Animal Care and Use Committee of the Hamamatsu University School of Medicine and were carried out in accordance with the approved guidelines. Human angiotensin II and bradykinin, used as calibration standard peptides, were purchased from Sigma-Aldrich Japan (Meguro, Japan). 9AA, used as a matrix, was purchased from Merck Millipore (Darmstadt, Germany).
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7

Cardiac Tissue Models for MI and Angiotensin II-Induced Cardiomyopathy

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Cardiac tissue for myocardial infarction (MI) and angiotensin II-treated (Ang II) cardiomyopathy models were performed as described previously [15 (link),16 (link)]. For the MI model, permanent left anterior descending coronary artery ligation was performed. For the Ang II model, human angiotensin II (Sigma-Aldrich) was administered to mice subcutaneously by osmotic pump (2mg/kg/day). MEF2A knockout mice were generated as previously described [17 (link)]. C57BL/10ScSn-mdx/J (mdx) mice were purchased from The Jackson Laboratory. Laminin-α2-knockout (DyW) tissue have been described previously [18 (link)].
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8

Preparation of Peptide Standards for Mass Spectrometry

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1 mg/mL stock solutions of each peptide (human angiotensin I, human angiotensin II, bradykinin, fibrinopeptide A, kemptide, neurotensin, porcine angiotensinogen, and substance P, all purchased from Sigma-Aldrich) were prepared in 0.1% formic acid (FA, Sigma-Aldrich, St. Louis, MO) in 10% acetonitrile (ACN, Fisher Scientific, Pittsburgh, PA) and deionized water. (Barnstead Nanopure Infinity System, Dubuque, IA). A peptide mixture containing 10 μM of each peptide was prepared from the stock solution in the same solvent mixture. Prior to MS analysis the peptide mixture solutions with final concentrations of 1 μM and 100 nM for each peptide were prepared by further dilution.
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9

Kinase Assay with Chemotaxis Proteins

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Human angiotensin II (#A9525), bradykinin (#B3259), diethylpyrocarbonate (DEPC) (#D5758), imidazole (#I5513), iodoacetamide (#I6125), and trypsin from bovine pancreas (#T1426) were all purchased from Sigma-Aldrich (St. Louis, MO). Urea (#424581000) was bought from Acros Organics (Fair Lawn, NJ). Formic acid (#A117), water (#W7), and acetonitrile (#A998) were obtained from Fisher Scientific (Fair Lawn, NJ). Amicon® centrifugal filters (#UFC501096 & #UFC800324) were from Millipore Sigma (Burlington, MA). A kinase buffer (50 mM K2HPO4, 5 mM MgCl2, 75 mM KCl, pH 7.5) and large unilamellar vesicles containing 60% 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 40% nickel chelating lipid DGS-Ni2+-NTA (1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)-iminodiacetic acid]succinyl}) were prepared using previously described methods.39 (link) CheA, CheW, and the cytoplasmic fragment (CF4Q) were expressed and purified using previously described methods.39 (link)
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10

Pharmacological Modulation of the Renin-Angiotensin System

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Human angiotensin II, irbesartan, losartan, PD123319, and everolimus were purchased from Sigma (Sigma Aldrich, St Louis, MO, USA).
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