Increasing the flexibility of the LANCE cAMP detection kit.

Abstract

The detection of cAMP signalling is a common endpoint in the study of G-protein coupled receptors. A number of commercially available kits enable easy detection of cAMP. These kits are based on competition for a cAMP binding site on an antibody or cAMP binding protein and as such have a limited dynamic range. Here, we describe the optimisation of the commercially-available LANCE cAMP detection kit (PerkinElmer) to enable detection in cell lysates. This kit has been designed for use with live cells, with detection reagents applied to cells without wash steps. The standard protocol therefore requires that all assay reagents are compatible with the antibody and the final fluorescent detection stage, limiting the range of assay media and test compounds that can be utilised. The entire experiment must be repeated if cAMP levels fall outside the limited dynamic range. Here we describe a modified protocol that enables the assay to be performed on cell lysates, thereby overcoming these limitations. In this modified protocol, cells are stimulated for a cAMP response in standard media/buffers, washed and then lysed. The cell lysate is then assayed using a modified protocol for the LANCE cAMP detection kit. Samples were tested for stability following a freeze-thaw cycle. The modified LANCE cAMP detection protocol gives a reproducible measurement of cAMP in cell lysate. Lysate samples remain stable when stored at -80°C. Separating the stimulation and detection phases of this cAMP assay allows a vast array of cell stimulation conditions to be tested. The lysate-modified protocol for the LANCE cAMP detection kit therefore increases the flexibility, versatility and convenience of the assay. As samples are insensitive to freeze-thaw, it enables retesting of samples under different dilution conditions to ensure that all samples remain within the dynamic range of the standard curve.