CatchPoint cGMP Fluorescent Assay Kit

Reagents for cyclic-GMP Detection

 

Improving the sensitivity and convenience of cGMP assays More sensitive and less susceptible to interferences than homogeneous assays Requires only a single wash step Rapid signal development (< 10 min) Flexible read time, compatible with high throughput screening (HTS) Convenient, no termination step needed Detection limit: 24 fmol/well for 96-well format Detection limit: 12 fmol/well for 384-well format

Introduction

The CatchPoint® cyclic-GMP fluorescent assay kit measures cGMP levels and guanylate cyclase activity via a competitive immunoassay format. The assay's high affinity reagents are optimized for convenience with high sensitivity and precision. This is particularly important in applications where cGMP levels are low. The cyclic-GMP fluorescent assay kit was especially developed for such conditions. For efficiency and economy, the reagents can be purchased as a kit for assay evaluation and in bulk for high-throughput screening. The assay is ideally suited for cell-based assays.

Sensitive

Many standard assays fail to produce viable data when cGMP levels are low unless acetylation is used. Screening agonists / antagonists of guanylate cyclase can be particularly problematic. The CatchPoint cGMP Assay Kit's single wash step significantly helps increase resolution, convenience, and improve data quality in these situations. In contrast to homogeneous formats, the assay is very resistant to interference from colored or fluorescent compounds. The compounds are washed away prior to the development step, and due to the fluorescent signal's intensity, small traces of residual fluorescence resulting from interference are insignificant.

	96-Well Format	384-Well Format
Detection Limit:	0.2 nM (24 fmol/well)	0.2 nM (12 fmol/well)
Maximum Measurable Concentration:	120 nM (14 pmol/well)	500 nM (30 pmol/well)
EC50	2.5 ± 0.6 nM (300 fmol)	2.9 ± 0.7 nM (170 fmol)

Convenient

With only a single wash step, the CatchPoint cGMP Assay Kit is easy to use. There are not multiple washes or radioactive components that are associated with other commercially available kits. The protocol is straightforward and easy to use. The assay can be run on the Analyst®, Gemini XS, Gemini EM or FlexStation™ Systems, further increasing the options for the user.

Flexible read time

In addition, the CatchPoint cGMP Fluorescent Assay Kit generates a fluorescent readout that is stable and capable of high precision, while not requiring exact timing in its incubation stages. No termination step is needed to halt the assay. This means the assay can be read in as little as 10 minutes or up to 24 hours after substrate addition. For this reason the assay is particularly suitable for robotic or workstation procedures.

Assay performance, 96-well data

Calibration curve: the calibration curve for the CatchPoint cyclic-GMP kit, read on the Analyst. Data was taken 60 minutes after addition of Stoplight Red substrate. The EC₅₀ of the cGMP calibration curve was 2.2 nM (264 fmol). Minimum detectable concentration was 0.2 nM (24 fmol).

		Figure 1. cGMP standard curve, 96-well data.

Atrial natriuretic peptide dose response in RFL-6 cells: a confluent monolayer of RFL-6 cells in a 12-well plate were pre-incubated for 10 minutes in Krebs Ringer bicarbonate buffer containing the non-selective phosphodiesterase inhibitor IBMX. Cells were subsequently stimulated with ANP for 15 minutes, lysed, and analyzed using the cGMP Fluorescent Assay Kit.

		Figure 2. Atrial natriuretic peptide dose response curve.

Assay performance, 384-well data

Calibration curve: calibration curve for the CatchPoint cyclic-GMP kit, read on the Analyst. Data was taken 60 minutes after addition of Stoplight Red substrate. EC50 of cGMP calibration curve was 3.4 nM (204 fmol). Minimum detectable concentration was 0.2 nM (12 fmol).

		Figure 3. cGMP standard curve, 384-well data.

Ordering Information

Product Number	R8074	R8065
Product Name	CatchPoint® cGMP 96-well Explorer Fluorescent Assay Kit	CatchPoint® cGMP 384-well Explorer Fluorescent Assay Kit
Unit Size	1 Explorer Kit provides: 2, 96-well black clear-bottom plates 100 mL Assay Buffer 1 vial Rabbit Anti-cGMP Antibody 1 vial cGMP Calibrator 1 vial HRP-cGMP Conjugate 100mL, 10X Wash Solution Concentrate 50 mL Cell Lysis Buffer 1 vial 100X Substrate 100 mL Substrate Buffer	1 Explorer Kit provides: 2, 384-well black clear-bottom plates 100 mL Assay Buffer 2 vials Rabbit Anti-cGMP Antibody 2 vials cGMP Calibrator 2 vials HRP-cGMP Conjugate 100mL, 10X Wash Solution Concentrate 50 mL Cell Lysis Buffer 1 vial 100X Substrate 100 mL Substrate Buffer
Product Number	R8075	R8066
Product Name	CatchPoint® cGMP 96-well Bulk Fluorescent Assay Kit	CatchPoint® cGMP 384-well Bulk Fluorescent Assay Kit
Unit Size	1 Bulk Kit provides: 10, 96-well black clear-bottom plates 100 mL Assay Buffer 2 vials Rabbit Anti-cGMP Antibody 2 vialss cGMP Calibrator 2 vials HRP-cGMP Conjugate 950 mL, 10X Wash Solution Concentrate 450 mL Cell Lysis Buffer 2 x 900 µL100X Substrate 950 mL Substrate Buffer	1 Bulk Kit provides: 20, 384-well black clear-bottom plates 2 x 950 mL cGMP Assay Buffer 2 vials Rabbit Anti-cGMP Antibody (lyophilized) 2 vials cGMP Calibrator 2 vials HRP-cGMP Conjugate 950 mL, 10X Wash Solution Concentrate 2 x 450 mL Cell Lysis Buffer 5 x 900 µL100X Substrate 950 mL Substrate Buffer

To place an order, call your local Molecular Devices Office or Molecular Devices Distributor.