I am doing Elisa using Alkaline Phosphatase and Pnpp. Pnpp is in powder form, I have made pnpp solution in a concentration of 10mg/10ml using phosphate buffer..
But the problem is I am not getting result after 30 min incubation of adding pnpp as substrate..it's taking long time more than 5/6 hrs to develop yellow colour for positive samples...why it's is happening..please help me
If the Pnpp solution will be prepared just before the use in ELISA, may resolve the problem. The longer storage of substrate may hamper it's actual activity.
Dissolve pNPP in either 0.1 M glycine buffer containing 1 mM MgCl2, 1 mM ZnCl2, pH 10.4, or 1 M diethanolamine buffer containing 0.5 mM MgCl2, pH 9.8, to desired concentration (typically a pNPP concentration of 1 mg/ml is used).
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