Aar 013

Manufactured by Alomone

Sourced in Israel

AAR-013 is a laboratory reagent produced by Alomone. It is used for research purposes in scientific settings. The core function of this product is to serve as a biochemical tool for experiments and investigations. Further details about its intended use or applications are not provided to maintain an unbiased and factual approach.

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Lab products found in correlation

8 protocols using aar 013

Abdominal Aortic Protein Expression Analysis

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Abdominal aortic segments were removed and stored at −80°C for analyses of
protein expression. The proteins from abdominal aorta were isolated with lysis
buffer (pH 8.0, 50 mmol/L tris–base, 100 mmol/L NaCl, 5 mmol/L EGTA,
Na₄P₂O₇ 50 mmol/L, MgCl₂ 1 M/L,
Nonidet p/40 1%, 0.3% triton X-100, sodium deoxycholate 0.5% and 1% protease
inhibitor cocktail). For the Western blotting experiments, 50 µg of each protein
sample was boiled and denatured in loading buffer containing 5%
2β-mercaptoethanol (Invitrogen, Waltham, MA, USA).Samples were separated by
electrophoresis on a 10% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE)
and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were
probed with diluted antibodies specific to Mas receptor (1:500; AAR013; Alomone
Labs, Jerusalem, Israel), Catalase (1:1000. sc271803, Santa Cruz Biotechnology,
Dallas, TX, USA), superoxide dismutase 2 (SOD-2) (1:1000, sc137254, Santa Cruz
Biotechnology, Dallas, TX, USA), NRF-2 (1:500, ab31163, Abcam, Cambridge, UK)
and NOX2/gp91 (1:500, sc74514, Santa Cruz Biotechnology, Dallas, TX, USA). Pixel
density was normalized to the expression of the reference protein glyceraldehyde
3-phosphate dehydrogenase (GAPDH, 1:1000, Cell Signaling Technology, Danvers,
MA, USA; #97166).

Costa-Fraga F.P., Goncalves G.K., Souza-Neto F.P., Reis A.M., Capettini L.A., Santos R.A., Fraga-Silva R.A., Stergiopulos N, & da Silva R.F. (2018). Age-related changes in vascular responses to angiotensin-(1-7) in female mice. Journal of the Renin-Angiotensin-Aldosterone System: JRAAS, 19(3), 1470320318789332.

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Immunohistochemical Localization of Angiotensin Receptors

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We used antibodies against Ang(1–7) Mas receptor (AAR 013, Alomone Labs Ltd, Jerusalem, Israel) and Ang-I(1–7)/AngII(1–7) (H-002-24, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA). The antigen reactivity of both agents has been confirmed for humans but also for the rat (data not shown). For immunohistochemistry we used 4 μm-thick paraffin sections that were cut onto SuperFrost plus microscopic slides (Menzel-Gläser, Gerhard Menzel GmbH, Braunschweig, Germany). The sections were dried at +60 °C for 1 h. Antigen retrieval was performed on re-hydrated sections in a microwave oven at 850 W twice for 7 min using 10 mM Tris 1 mM EDTA retrieval buffer (pH 9.0) as retrieval solution.

Vaajanen A., Kalesnykas G., Vapaatalo H, & Uusitalo H. (2015). The expression of Mas-receptor of the renin–angiotensin system in the human eye. Graefe's Archive for Clinical and Experimental Ophthalmology, 253(7), 1053-1059.

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Protein Expression Analysis Protocol

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Tissues were dissolved in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, protease and phosphatase inhibitor mixture (Roche Diagnostics)). Protein concentrations were determined using a BCA assay kit (Pierce Diagnostics). Protein was separated by 10% (wt/vol) SDS/PAGE, transferred to a PVDF membrane (Millipore), blocked in 5% (wt/vol) skim milk in TBST (0.02 M Trisbase, 0.14 M Vehicle, 0.1% Tween 20, pH 7.4), and incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies conjugated with HRP. The following primary antibodies were used: anti-UCP1 (ab10983, Abcam), anti-PGC1ɑ (ab54481, Abcam), anti-OXPHOS (ab110413, Abcam), anti-Mas1 (AAR-013, Alomone labs), anti-Akt (#9272, cell signaling technology), anti-p-Akt308 (#13038, cell signaling technology), anti-FoxO1 (#2880, cell signaling technology), anti-p-FoxO1 (#84192, cell signaling technology), anti-PKA (#4782, cell signaling technology), anti-p-PKA (#9621, cell signaling technology), anti-ACE2 (#92485, cell signaling technology), and actin (#4970, Cell Signaling Technology). Signals were detected with Super Signal West Pico Chemiluminescent Substrate (Pierce).

Cao X., Shi T.T., Zhang C.H., Jin W.Z., Song L.N., Zhang Y.C., Liu J.Y., Yang F.Y., Rotimi C.N., Xu A, & Yang J.K. (2022). ACE2 pathway regulates thermogenesis and energy metabolism. eLife, 11, e72266.

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Subcellular Localization of Mas Receptor

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In order to study the presence of Mas receptor at mitochondrial and nuclear level, different cell line cultures were grown on glass coverslips and incubated with the fluorescent probes for mitochondria or nuclei. MitoTracker Deep Red (MTDR; 20 nM; Molecular Probes) was used for mitochondrial labeling and the DNA-binding dye Hoechst 33342 for nuclear labeling. After labeling, the cells were fixed and immunoreacted for the rabbit polyclonal Mas receptor antibody (AAR-013, Alomone, 1:100).

Costa-Besada M.A., Valenzuela R., Garrido-Gil P., Villar-Cheda B., Parga J.A., Lanciego J.L, & Labandeira-Garcia J.L. (2017). Paracrine and Intracrine Angiotensin 1-7/Mas Receptor Axis in the Substantia Nigra of Rodents, Monkeys, and Humans. Molecular Neurobiology, 55(7), 5847-5867.

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Western Blot Analysis of ACE2 and RAS Receptors

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Homogenates from rat lung tissue or A549 cells were lysed in RIPA buffer containing PMSF (Sigma) and protease inhibitor cocktail (Sigma). Total proteins were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). An equal amount of protein lysates or cell culture medium was separated on 8–10% Bis-Tris polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were incubated overnight at 4°C with primary antibodies against the extracellular domain of ACE2 (ab108252; Abcam; 1:1000), AT1 (sc-31181; Santa Cruz; 1:200), AT2 (sc-9040; Santa Cruz; 1:200) and Mas (AAR-013; Alomone labs; 1:1000) receptors. Membranes were reincubated with loading controls: anti-α-tubulin (T5168; 1:50.000; Sigma) or GAPDH (G9545; 1:25.000; Sigma), or ponceau (Sigma, P7170-1L). The following horseradish peroxidase (HRP)-conjugated secondary antibodies were used: goat anti-rabbit-HRP and goat anti-mouse-HRP (Santa Cruz Biotechnology; 1:2500). Bound antibody was detected with an Immun-Star HRP Chemiluminescent Kit (Bio-Rad; 170-5044) and visualized with a chemiluminescence detection system (Bio-Rad; Molecular Imager ChemiDoc XRS System). The data were then expressed relative to the value obtained for the control to counteract possible variability among batches.

Pedrosa M.A., Valenzuela R., Garrido-Gil P., Labandeira C.M., Navarro G., Franco R., Labandeira-Garcia J.L, & Rodriguez-Perez A.I. (2021). Experimental data using candesartan and captopril indicate no double-edged sword effect in COVID-19. Clinical Science (London, England : 1979), 135(3), 465-481.

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Validating commercial antibodies for MasR

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Four commercial antibodies raised against different domains of the MasR were selected for their validation. Two antibodies (sc-135063 and sc-54682) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); AAR-013 was from Alomone Labs (Jerusalem, Israel) and NLS1-1531 was from Novus Biologicals (Littleton, CO, USA). During the last phase of this study Santa Cruz Biotechnology discontinued a large number of its polyclonal products including sc-135063 and sc-54682 antibodies. The information of immunogen used, reactivity and applications as provided by the manufacturers is presented in Fig 1.

Burghi V., Fernández N.C., Gándola Y.B., Piazza V.G., Quiroga D.T., Guilhen Mario É., Felix Braga J., Bader M., Santos R.A., Dominici F.P, & Muñoz M.C. (2017). Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies. PLoS ONE, 12(8), e0183278.

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Western Blot Analysis of Mas Receptor and ACE2

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Tissue from rat and mouse ventral midbrain was lysed in RIPA buffer containing protease inhibitor cocktail (Sigma) and PMSF (Sigma). Lysates were centrifuged and proteins were quantified using the Pierce BCA Protein Assay kit (Thermo Scientific). Equal amount of protein lysates, isolated mitochondria, and nuclei was separated on a 10% Bis-Tris polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were incubated overnight with primary antibodies against the Mas receptor (AAR-013; 1:1000) from Alomone (see previous section for specificity) and angiotensin converting enzyme 2 (ACE2; ab108252; 1:1000) from Abcam. Blots were reprobed with anti-GAPDH (G9545; 1:50,000, Sigma) as a loading control. The membranes were incubated with the following HRP-conjugated secondary antibodies: goat anti-rabbit (1:2500) and goat anti-mouse (1:2500) from Santa Cruz Biotechnology. Immunoreactive bands were detected with an Immun-Star HRP Chemiluminescent Kit (170-5044; Bio-Rad) and visualized with a chemiluminescence detection system (Molecular Imager ChemiDoc XRS System; Bio-Rad). The data were then expressed relative to the value obtained for the control (100%) to counteract possible variability among batches. Finally, the results were expressed as means ± SEM.

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Immunostaining of AT1R and Mas Receptors

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For immunostaining, the tissues were embedded with OCT compound and rapidly frozen in isopropanol-chilled liquid nitrogen. They were stored at −80°C until 7 µm sections were made using a cryostat. The primary antibody for detection of AT₁R-immunoreactivity was a rabbit polyclonal antibody to the AT₁R (1:100 dilution; catalog no. sc-1173, Santa Cruz). Mas-immunoreactivity was detected using anti-Mas antibody (1:60 dilution; AAR-013 Alomone Labs). The sections were incubated with a secondary antibody, Alexa Fluor 488 goat, anti-rabbit IgG and DAPI to counterstain cell nuclei. Immunostaining was visualized with a confocal microscope (Zeiss 710).

Sabharwal R., Cicha M.Z., Sinisterra R.D., de Sousa F.B., Santos R.A, & Chapleau M.W. (2014). CHRONIC ORAL ADMINISTRATION OF ANG-(1-7) IMPROVES SKELETAL MUSCLE, AUTONOMIC, AND LOCOMOTOR PHENOTYPES IN MUSCULAR DYSTROPHY. Clinical science (London, England : 1979), 127(2), 101-109.

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