After chemical lysis of the cells in lysis buffer (0.5% TritonX-100, 20 mM Tris pH 7.4, 10% Sucrose) NEP activity assay was performed according to Miners et al. (2008b (link)) with minor modifications utilizing the anti-NEP antibody AF1182 (R&D Systems, Minneapolis, Minn., USA) and 5 μM MCA-RPPGFSAFK(DNP)-OH fluorogenic peptide substrate (R&D Systems, Minneapolis, Minn., USA). Fluorescence was measured with an excitation wavelength of 320 ± 10 nm and an emission wavelength of 405 ± 10 nm in a Safire2 Fluorometer (Tecan, Crailsheim, Germany) as described above. Unspecificity of the assay was 22.1% (± 2.4%, p ≤ 0.001) as measured in presence of 10 μM thiorphan (Santa Cruz Biotechnology, Dallas, USA) (Supplementary Figure 1A ).
Af1182
Manufactured by R&D Systems
Sourced in United States
AF1182 is a recombinant human protein that functions as an enzyme. It is a member of the serine protease family and is involved in the regulation of biological processes.
Automatically generated - may contain errors
4 protocols using af1182
1
Fluorometric NEP Activity Assay
2
Quantitative NEP Activity Assay
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Measurement of NEP activity was performed as published in Miners et al. [84 (link)], with minor modifications utilizing the anti-NEP antibody AF1182 (R&D Systems, Minneapolis, MN, USA) and 5 μM MCA-RPPGFSAFK(DNP)-OH fluorogenic peptide substrate (R&D Systems, Minneapolis, MN, USA). Cells were chemical lysed in lysis buffer containing 0.5% TritonX-100, 20 nM Tris pH 7.4, 10% sucrose and fluorescence at an excitation wavelength of 320 ± 10 nm, and an emission wavelength of 405 ± 10 nm was measured in a Safire2 Fluorometer (Tecan, Crailsheim, Germany).
3
Fluorometric Assay of Neprilysin Activity
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The neurons and hippocampal tissues of mice from different groups were placed into a mixed cleavage buffer comprising 0.5% Triton X-100 (Biyuntian Co., Ltd., ST795, China), 89.5% 20 mM Tris-HCl (pH 7.4, Carnoss Technology Co., Ltd., Kns201612012110, China), and 10% sucrose and incubated at 4°C for 25 min. After incubation, the sample was placed in a mixture of 2 μl anti-NEP antibody (1 : 500, R&D Systems, AF1182, USA), 5 μM MCA RPPGFSAFK (DNP) OH fluorescent peptide substrate (R&D Systems, ES005, USA), and HEPES buffer (pH 7.4, Invitrogen, 28398, USA) and incubated at room temperature for 30 min. The OD of the samples in each group was measured at an excitation wavelength of 320 ± 10 nm and emission wavelength of 405 ± 10 nm using iMark fluorimeter and microplate reader (Bio Rad, V111584, USA).
4
Western Blot Analysis of Neuronal Proteins
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Adherent primary neurons were collected using radioimmunoprecipitation assay buffer, whereas the tissue homogenates of cortical or hippocampal tissues of mice were dissolved in protein lysate (Biyuntian Co., Ltd., P0013C, China). Centrifugation was performed (12000 rpm, 15 min, 4°C), and the supernatant was absorbed as extracted protein.
In all, 40 μg of protein was extracted from each sample and isolated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was placed in a blocking solution to reduce nonspecific binding. Subsequently, the membrane was washed with Tris-buffered saline Tween-20 (TBST).
Following this, the membrane was incubated with GAPDH antibody (1 : 1000, Abcam, Ab9485, UK), APP antibody (1 : 1000, Abcam, Ab241592, UK), Navβ2 antibody (1 : 1000, Abcam, Ab138064, UK), BDNF antibody (1 : 1000, Abcam, Ab108319, UK), or NEP antibody (1 : 1000, R&D Systems, AF1182, USA) at 4°C overnight. After washing with 1x TBST, the membrane was incubated with secondary antibodies (1 : 1000, Abbkine, A23220/A23410, USA) at room temperature for 1 h and rinsed with 1x TBST again. All results were photographed using Bio-Rad Gel Imaging System (ChemiDoc™ XRS+). ImageJ (National Institutes of Health, USA) was used for strip quantitative analysis.
In all, 40 μg of protein was extracted from each sample and isolated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was placed in a blocking solution to reduce nonspecific binding. Subsequently, the membrane was washed with Tris-buffered saline Tween-20 (TBST).
Following this, the membrane was incubated with GAPDH antibody (1 : 1000, Abcam, Ab9485, UK), APP antibody (1 : 1000, Abcam, Ab241592, UK), Navβ2 antibody (1 : 1000, Abcam, Ab138064, UK), BDNF antibody (1 : 1000, Abcam, Ab108319, UK), or NEP antibody (1 : 1000, R&D Systems, AF1182, USA) at 4°C overnight. After washing with 1x TBST, the membrane was incubated with secondary antibodies (1 : 1000, Abbkine, A23220/A23410, USA) at room temperature for 1 h and rinsed with 1x TBST again. All results were photographed using Bio-Rad Gel Imaging System (ChemiDoc™ XRS+). ImageJ (National Institutes of Health, USA) was used for strip quantitative analysis.
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