Cyclic AMP and GMP XP® Assay Kits
Cell Signaling Technology® now offers Cyclic AMP and Cyclic GMP XP® Assay Kits (#4339 and #4360) which are based on the competition immunoassay principle, and can be used to measure the activation of many G protein coupled receptors (GPCRs). In these kits cyclic nucleotide in the sample of interest competes with a fixed amount of cyclic nucleotide-HRP conjugate provided in the kit for the binding to cyclic nucleotide-specific XP® rabbit monoclonal antibody that is pre-coated on the assay plate. Because of the competitive nature of this assay, the magnitude of the absorbance is inversely proportional to the quantity of cyclic nucleotide in the sample.
Cyclic GMP XP® Assay Kit #4360
Cyclic Nucleotide EIA Kits
|8020||Cyclic GMP XP® Chemiluminescent Assay Kit|
|4360||Cyclic GMP XP® Assay Kit|
|8019||Cyclic AMP XP® Chemiluminescent Assay Kit|
|4339||Cyclic AMP XP® Assay Kit|
Figure 1. Cell-based assay: Treatment of RFL-6 cells with sodium nitroprusside (SNP) increases cGMP concentration as detected by Cyclic GMP XP® Assay Kit #4360. RFL-6 cells were seeded at 2x105 cells/well in a 12-well plate and incubated overnight. Cells were either left untreated or pretreated with 0.5 mM IBMX for 15 minutes prior to SNP treatment (30 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follows: % activity=100x[(A-Abasal)/(Amax-Abasal)], where A is the sample absorbance, Amax is the absorbance at maximum stimulation (i.e., high SNP concentration), and Abasal is the absorbance at basal level (no SNP). SNP is a nitric oxide donor that directly activates soluble guanylyl cyclases and increases cellular cGMP concentration. IBMX is a non-specific inhibitor of cAMP and cGMP phosphodiesterases that promotes accumulation of cAMP and cGMP in cells.
Cyclic AMP XP® Assay Kit #4339
Figure 2. Cell-based assay: Treatment of 293 cells with isoproterenol increases the cAMP concentration as detected by Cyclic AMP XP® Assay Kit #4339. 293 cells were seeded at 3x104 cells/well in a 96-well plate and incubated overnight. Cells were pretreated with 0.5 mM IBMX for 30 minutes prior to isoproterenol treatment (3 minutes) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and percentage of activity (right) are shown above. The percentage of activity is calculated as follow: % activity=100*[(A-Abasal)/(Amax-Abasal)], where A is the absorbance of the sample, Amax is the absorbance at maximum stimulation (i.e., high isoproterenol concentration), and Abasal is the absorbance at basal level (no isoproterenol). Isopropterenol is a β-adrenoceptor agonist and activates beta-2 adrenergic receptors (ADRB2) that are endogenously expressed on 293 cells. Activation of ADRB2 then leads to activation of adenylyl cyclase and synthesis of cAMP as its second messenger.