We tried to measure cAMP from a mouse heart using ELISA. The problem is that we are not getting stable data from varied mice hearts sample. As cAMP can be degraded easily, we suspect that there might be degradation of cAMP while harvesting/preparing heart lysate. Any suggestions?
dear Sailesh.
Maybe an option for you will be measurement of phosphodiesterase activity or/and adenylate cyclase activity in your samples.
Sailesh,
If you are using cells means, you can try adding PDE inhibitor, which prevents the degradation of cAMP.. In case of tissue, suspend your tissue lysate in 0.1M HCl, this also prevents cAMP degradation. Protease activity can affect the ELISA Ab.
Dear all,
We successfully measured cAMP in the mice heart sample. We used 0.1 N HCl for homogenization. The supernatant was neutralized by the addition of 0.1 N NaOH, heated at 70 degree celcius, centrifuged again, and then diluted with an equal volume of diluent, and measured by ELISA.
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