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You need to replace the high speed centrifugation suggested in the Calcineurin cellular activity assay kit (BML-AK816) manual which pellets the nuclei with a low speed spin to clarify the samples from large debris. You will also need to make sure that the nuclear membrane is being broken. For some cells, the provided lysis buffer would be sufficient to lyse the cells and break open the nuclei. However, this may not be true for all cell types. If you wish to accurately measure nuclear as well as cytosolic calcineurin you will need to optimize sample preparation and add appropriate controls to make sure the nucleus is lysing in your case. Please note that any lysis buffers used with this assay will need to be free of free phosphate contamination. Also high concentrations of detergents can interfere with the function of BIOMOL Green either by slowing the color development reaction or by interfering with the OD readings themselves. We do not recommend going above 0.1% triton X-100, 0.05% NP40 or 0.01% Brij 35. Tween detergents are not recommended.