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Amplite® Fluorimetric Renin Assay Kit *Red Fluorescence*

Renin is an enzyme that participates in the body's renin-angiotensin system (RAS) that mediates extracellular volume and arterial vasoconstriction. It regulates blood pressure and electrolyte homoeostasis. Angiotensin II constricts blood vessels leading to increased blood pressure. It also increases the secretion of ADH and aldosterone, and stimulates the hypothalamus to activate the thirst reflex. An over-active renin-angiotension system leads to vasoconstriction and retention of sodium and water. Renin has been identified to be an attractive target for the treatment of hypertension. The Amplite® Renin Assay Kit provides a convenient assay for high throughput screening of renin inhibitors and renin activity using our proprietary Tide Fluor™ 3 (TF3)/Tide Quencher™ 3 (TQ3) fluorescence resonance energy transfer (FRET) peptide. In the FRET peptide, the fluorescence of TF3 is quenched by TQ3. Upon cleavage into two separate fragments by renin, the fluorescence of TF3 is recovered, and the fluorescent signal can be easily monitored by a fluorescence microplate reader. This assay is about fifty fold more sensitive than an EDANS/DABCYL-based assay. With the Amplite® Renin Assay Kit, we have detected as little as 1ng renin in a 100 µL reaction volume. However, the selectivity of the rennin substrate used in the kit has not been thoroughly tested. It may also response to other proteases since peptide-based protease substrates generally have low selectivity.

Example protocol

AT A GLANCE

Protocol Summary
  1. Prepare and add Renin standard and test samples (50 µL)

  2. Add Renin Red™ substrate working solution (50 µL)
  3. Incubate for 30 - 60 min at 37 °C incubator (for end point reading)
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important     Thaw all the kit components at room temperature before starting the experiment. Prepare Renin containing biological samples as desired.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13530

Renin standard
Add 12.5 µL of 40 µg/mL Renin Standard (Component B) into 487.5 µL of Assay Buffer (Component C) to get 1000 ng/mL Renin standard solution (Ren7). Take 150 µL of 1000 ng/mL Renin standard solution to perform 1:3 serial dilutions to get serially diluted Renin standards (Ren6 - Ren1).

PREPARATION OF WORKING SOLUTION

Add 50 μL of Renin Red™ Substrate (Component A) into 5 mL of Assay Buffer (Component C) to make a total volume of 5.05 mL.

Note        The Renin Red™ Substrate should be used promptly.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of Renin standards and test samples in a solid black 96-well microplate. Ren = Renin Standards (Ren1-Ren7, 1 to 1000 ng/mL); BL = Blank Control; TS = Test Samples.

BLBLTSTS
Ren1Ren1......
Ren2Ren2......
Ren3Ren3
Ren4Ren4
Ren5Ren5
Ren6Ren6
Ren7Ren7

Table 2. Reagent composition for each well.

Note     The Renin standards are for positive control only, and should not be relied on as a quantitation standard for enzyme activity.

WellVolumeReagent
Ren1 - Ren750 µLserial dilution (1 to 1000 ng/mL)
BL50 µL

Assay Buffer (Component C)

TS50 µLsample
  1. Prepare Renin containing biological samples as desired.
  2. Prepare the Renin standards and/or Renin-containing test samples according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
  3. Pre-incubate the plate at a desired temperature for the enzyme reaction (e.g. 25 °C or 37 °C) for 10 - 15 min, if you are screening Renin inhibitors.
  4. Add 50 µL (96-well) or 25 µL (384-well) of Renin Red™ substrate working solution to the Standard sample, control  and test wells of the assay plate.

  5. Incubate the reaction at 37 °C incubator for 30 to 60 minutes.
  6. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em = 540/590 nm (cut off = 570 mn).

    Note     The selectivity of the renin substrate used in the kit has not been thoroughly tested. It may also respond to other proteases since peptide-based protease substrates generally have low selectivity. One might use a renin-specific inhibitor for its specific test, such as in the presence of a renin-specific inhibitor, hydrolysis of the substrate is only due to the non-specific protease activity. The difference between the total activity and the activity in the presence of renin specific Inhibitor gives the renin activity in the sample.

    For kinetic reading: Immediately start measuring fluorescence intensity and continuously record data every 5 minutes for 30 to 60 minutes.

    For end-point reading: Incubate the reaction at 37 °C for 60 minutes or longer, kept from light if possible. And then measure the fluorescence intensity.

Spectrum

Citations

View all 1 citations: Citation Explorer
Influence of Dinitrosyl Iron Complexes on the Activity of Enzymes, Indicators of Cardiovascular Diseases
Authors: Akentieva, Natalia and Sanina, Natalia and Gizatullin, Artur and Shkondina, Natalia and Andreeva, Anna and Shram, Stanislav and Aldoshin, Sergei
Journal: (2022)

References

View all 70 references: Citation Explorer
Activation of the succinate receptor GPR91 in macula densa cells causes renin release
Authors: Vargas SL, Toma I, Kang JJ, Meer EJ, Peti-Peterdi J.
Journal: J Am Soc Nephrol (2009): 1002
Hypotonicity-induced Renin exocytosis from juxtaglomerular cells requires aquaporin-1 and cyclooxygenase-2
Authors: Friis UG, Madsen K, Svenningsen P, Hansen PB, Gulaveerasingam A, Jorgensen F, Aalkjaer C, Skott O, Jensen BL.
Journal: J Am Soc Nephrol (2009): 2154
Cytosolic renin is targeted to mitochondria and induces apoptosis in H9c2 rat cardiomyoblasts
Authors: Wanka H, Kessler N, Ellmer J, Endlich N, Peters BS, Clausmeyer S, Peters J.
Journal: J Cell Mol Med (2009): 2926
Measurement of plasma renin concentration in cats by use of a fluorescence resonance energy transfer peptide substrate of renin
Authors: Schmiedt CW, Hurley KA, Tong X, Rakhmanova VA, Po CL, Hurley DJ.
Journal: Am J Vet Res (2009): 1315
Prolonged exposure of cardiac cells to renin plus angiotensinogen reduces intracellular renin in the failing heart. On the role of angiotensin II-AT1 complex internalization
Authors: De Mello WC, Gerena Y.
Journal: Regul Pept (2009): 139
Page updated on October 8, 2024

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Catalog Number13530
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Spectral properties

Excitation (nm)

545

Emission (nm)

572

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components

Renin dose response was measured with Amplite® Fluorimetric Renin Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Renin dose response was measured with Amplite® Fluorimetric Renin Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).
Renin dose response was measured with Amplite® Fluorimetric Renin Assay Kit in a 96-well solid black plate using a Gemini fluorescence microplate reader (Molecular Devices).