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PI3K alpha biochemical assay

MM Marinela Méndez-Pertuz
PM Paula Martínez
CB Carmen Blanco-Aparicio
EG Elena Gómez-Casero
AG Ana Belen García
JM Jorge Martínez-Torrecuadrada
MP Marta Palafox
JC Javier Cortés
VS Violeta Serra
JP Joaquin Pastor
MB Maria A. Blasco
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The kinase activity was measured by using the commercial ADP HunterTM Plus assay available from DiscoveRx (#33-016), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, PI3K (p110α) was purchased from Cama Biosciences (#07CBS-0402A). The assay was performed following the manufacturer recommendations. To calculate the IC50 of the selected compounds, serial 1:5 dilutions were used in a concentration range from 128 pM to 50 μM fluorescence counts were read in a Victor instrument (Perkin Elmer) with the recommended settings (544 and 580 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for the enzyme (Le., 100 % PI3 kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose-response curve by using Activity base software from IDBS.

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