Amplite™ Fluorimetric Renin Assay Kit *Red Fluorescence*

! Interactive Product Finder: This product has alternative forms and/or upgrades. View now


Image Viewer
Analyze with Quest Graph™Plan a serial dilution
Close (X)
1001010010- Dose-responseData legend Generated with Quest Graph™ Renin (ng/mL) RFU Hover mouse to interact
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).
Loading...
 
Unit Size: Cat No: Price (USD): Qty:
100 tests 13530 $195


Export item/cart as Excel file

Send item/cart as email
EXPORT TO EXCEL X

Export:
EXPORT TO EMAIL X
Important: We request your email address to ensure that the recipient(s) knows you intended for them to see the email, and that it is not junk mail.
Export:
Your Name*:
Your Email*:
Recipient Email*:
Your Personal Message:
Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

PlatformsFluorescence microplate reader
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Peptidases and Proteases
Related
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 at Ex/Em = 540/590 nm. 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.




Spectrum Advanced Spectrum Viewer

Sorry, your browser does not support inline SVG. Relative Intensity (%) 100 80 60 40 20 0 Sorry, your browser does not support inline SVG.
Sorry, your browser does not support inline SVG. Sorry, your browser does not support inline SVG.
Move mouse over grid to display wavelength & intensity values.

300
400
500
600
700
800
900
Wavelength (nm)





Protocol


Quick Preview

This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Add appropriate controls, or 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 notes
Thaw all the kit components at room temperature before starting the experiment. Prepare Renin containing biological samples as desired.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
Preparation of standard solution
Renin standard

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

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.

BL BL TS TS
Ren1 Ren1 ... ...
Ren2 Ren2 ... ...
Ren3 Ren3    
Ren4 Ren4    
Ren5 Ren5    
Ren6 Ren6    
Ren7 Ren7    

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.

Well Volume Reagent
Ren1 - Ren7 50 µL serial dilution (1 to 1000 ng/mL)
BL 50 µL Assay Buffer (Component B)
TS 50 µL sample
  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 sample and control 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.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Renin samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator/

Figure 1. 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).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





Interactive Product Finder

Similar products
Product nameCurrent ProductRelated Product
Amplite™ Fluorimetric NAD Assay Kit *Blue Fluorescence*
Renin
Red
NAD
Blue
Amplite™ Fluorimetric NADP Assay Kit *Blue Fluorescence*
Renin
Red
NADP
Blue


Product nameCurrent ProductRelated Product
Amplite™ Fluorimetric Acetylcholine Assay Kit *Red Fluorescence*
Renin
Acetylcholine
Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Red Fluorescence*
Renin
Acetylcholinesterase
Amplite™ Fluorimetric Catalase Assay Kit *Red Fluorescence*
Renin
Catalase
See More (12)
Amplite™ Fluorimetric Acetylcholinesterase Assay Kit *Green Fluorescence*
Renin
Red
Acetylcholinesterase
Green
Amplite™ Fluorimetric NADH Assay Kit *Red Fluorescence*
Renin
NADH
Amplite™ Fluorimetric Glutathione Assay Kit *Green Fluorescence*
Renin
Red
Glutathione
Green
Amplite™ Fluorimetric NADPH Assay Kit *Red Fluorescence*
Renin
NADPH
Amplite™ Fluorimetric Myeloperoxidase Assay Kit *Red Fluorescence*
Renin
Myeloperoxidase
Amplite™ Fluorimetric Neuraminidase Assay Kit *Blue Fluorescence*
Renin
Red
Neuraminidase
Blue
Amplite™ Fluorimetric Sphingomyelin Assay Kit *Red Fluorescence*
Renin
Sphingomyelin
Amplite™ Fluorimetric Sphingomyelinase Assay Kit *Red Fluorescence*
Renin
Sphingomyelinase
Amplite™ Fluorimetric Glycerol Assay Kit
Renin
Red Fluorescence
Glycerol
Amplite™ Fluorimetric Xanthine Assay Kit
Renin
Red Fluorescence
Xanthine
Amplite™ Fluorimetric Pyruvate Assay Kit
Renin
Red Fluorescence
Pyruvate
Amplite™ Fluorimetric Oxaloacetate Assay Kit *Red Fluorescence*
Renin
Oxaloacetate






References

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

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

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

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

Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure
Authors: Hanner F, von Maltzahn J, Maxeiner S, Toma I, Sipos A, Kruger O, Willecke K, Peti-Peterdi J.
Journal: Am J Physiol Regul Integr Comp Physiol (2008): R371

Expression of functional human (pro)renin receptor in silkworm (Bombyx mori) larvae using BmMNPV bacmid
Authors: Du D, Kato T, Nabi AH, Suzuki F, Park EY.
Journal: Biotechnol Appl Biochem (2008): 195

Fluorescence activated cell sorting of transiently transfected As4.1 cells shows renin enhancer directs on/off switching of renin promoter in vitro
Authors: Morris BJ.
Journal: Clin Exp Pharmacol Physiol (2008): 367

Homocysteine, renin and aldosterone in patients with Cushing's syndrome
Authors: Zacharieva S, Kirilov G, Orbetzova M, Elenkova A, Shigarminova R, Lozanov V, Mitev V.
Journal: Methods Find Exp Clin Pharmacol (2008): 221

In vivo analysis of key elements within the renin regulatory region
Authors: Glenn ST, Jones CA, Pan L, Gross KW.
Journal: Physiol Genomics (2008): 243


View More Citations




Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Certificate of Analysis