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Cyclic-AMP & IP-One

GPCR second messenger assays

Following activation, GPCRs transmit information within cells via the modulation of two second messengers, cyclic AMP for Gi/s coupled receptors and inositol-triphosphate (IP3) for Gq coupled receptors.

While cAMP accumulates in the presence of a phosphodiesterase inhibitor (e.g. IBMX), IP3 is unstable and rapidly enters the metabolic inositol phosphate cascade. However, in the presence of lithium chloride, IP1, a metabolic product of IP3, accumulates and offers a viable biomarker for the visualization of Gq-coupled receptor activity.

Features

  • Homogeneous, non-radioactive assays
  • Fast, robust and cost efficient alternative to ELISA kits

Applications

  • Gs, Gi or Gq coupled receptor activation
  • Assess agonists, antagonists, allosteric modulators and inverse agonists
  • Detection of slow acting agonist in functional assay
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<p class="fr ml20"><img src="/sites/default/files/htrf/products/gpcr-signaling-pathways.jpg" style="width: 350px; height: 267px; " /></p>
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<p>These competitive immunoassays are based on an IP1 or cAMP specific monoclonal antibody, labeled with a Terbium-cryptate. IP1 or cAMP produced by cells compete with d2-labeled IP1 or cAMP for binding to the antibody. Thus, the HTRF signal is inversely proportional to the level of cellular IP1 or cAMP present.</p>
<p class="fl"><img src="/sites/default/files/htrf/products/ip-one-camp-cell-based-protocol.jpg" style="width: 260px; height: 186px; " /></p>
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<p><img src="/sites/default/files/htrf/products/ip-one-log-nm.gif" style="width: 250px; height: 186px; " /></p>
<p class="legend ml10">IP1 standard curve performed using protocol outlined in IP-One Tb assay product insert.</p>
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<table>
<thead>
<tr class="gris">
<th>&nbsp;</th>
<th>S/B standard curve</th>
<th>IC50</th>
<th>Specificity</th>
</tr>
</thead>
<tbody>
<tr>
<th>cAMP dynamic 2 kit</th>
<td>&ge;15</td>
<td>3 to 5.6 nM</td>
<td>No cross-reactivity with 500 nM ATP, ADP, cGMP</td>
</tr>
<tr class="gris">
<th>IP-One Tb kit</th>
<td>&ge;18</td>
<td>&le;140 nM</td>
<td>No cross-reactivity with 50 &micro;M Myo-inositol, PIP2, IP2, IP3, IP4 or PIP3</td>
</tr>
</tbody>
</table>





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