Renin enzymatic activity from EDTA-aprotinin supplemented mouse plasma samples (described in the Section 4.2 ) were measured in a 96-well microplate (Synergy HT reader and Gen5 v1.09 software, BioTek Instruments, Inc., Winooski, VT, USA) and quantified using exogenous fluorescence resonance transfer (FRET) peptide substrates of renin FRET-QXL™520/5-FAM, optimized for mouse renin (SensoLyte 520 mouse renin assay kit, AnaSpec, Fremont, CA, USA) as previously reported [25 (link),27 (link),32 (link),78 (link),79 (link),80 (link),81 (link)]. Cleavage of the FRET substrate by mouse renin results in the recovery of quenched fluorescence of 5-FAM, which was detected at excitation/emission = 490/520 nm with minimum autofluorescence of plasma samples. The 5-FAM fluorescent reference standard curve was used for results quantification. It is important to note that the plasma renin activity concentration assay differs from plasma renin activity (PRA) and active renin concentration (ARC)/ active plasma renin concentration (APRC) which have been historically used to report active renin in clinical trials [32 (link)].
Sensolyte 520 mouse renin assay kit
Manufactured by AnaSpec
Sourced in United States
The SensoLyte 520 mouse renin assay kit is a fluorescence-based assay designed to measure the activity of mouse renin. The kit provides all the necessary components, including the renin substrate, buffer, and a fluorogenic reporter. It enables the detection of renin activity in mouse samples, such as plasma, tissue homogenates, or cell lysates.
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4 protocols using sensolyte 520 mouse renin assay kit
1
Quantifying Mouse Plasma Renin Activity
2
Quantifying Murine Renin Activity
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Renin enzymatic activities in plasma samples were measured and quantified by cleavage of exogenous fluorescence resonance transfer (FRET) peptide substrates of renin FRET-QXL™520/5-FAM, optimized for mouse renin using a SensoLyte 520 mouse renin assay kit (AnaSpec, Fremont, CA, USA) as previously reported [35 (link),36 (link),37 (link),47 (link)].
3
Plasma Biomarkers Measurement Protocol
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Plasma corin, ANP (as N terminus-ANP), BNP (as C terminus-BNP), cGMP, angiotensin II and aldosterone levels were measured by enzyme immunoassays according to the manufacturer’s protocols (USCN Life Science Inc., China; Phoenix Pharmaceuticals, Inc.,Burlingame, CA; Enzo Life Science Inc.,Farmingdale, NY, Abcam Inc.,Cambridge, MA). Renin activities in plasma samples were measured by cleavage of a fluorescence resonance energy transfer substrate using a SensoLyte 520 mouse renin assay kit (AnaSpec, Fremont,CA) that allows the direct assessment of renin activity [34 (link)].
4
Fluorescence-Based Continuous Renin Assay
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The SensoLyte 520 Mouse Renin Assay kit (Anaspec, Fremont, California) was used to assay for renin activity. This kit allows for continuous assay of renin activity using a 5-FAM/QXL 520 fluorescence resonance energy transfer (FRET) peptide. In the FRET peptide, the fluorescence of 5-FAM is quenched by QXL 520. Upon cleavage into two separate fragments by mouse renin, the fluorescence of 5-FAM is recovered. This was measured with a fluorescence multiwell plate reader (Synergy HTX, Biotek, Winooski, Vermont) at excitation/emission: 〖"λ " 〗_ex = 485 nm / 〖"λ " 〗_em = 528 nm. Fluorescence readings are expressed in relative fluorescence units (RFU). Instruments were calibrated using a 5-FAM fluorescence reference standard. The plates were placed in the multiwell plate reader for 60 min, with measurements taken every 5 min.
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