How to calculate alkaline phosphatase activity from standard curve of its substrate?
Answers
Explanation:
You need to know the the extinction coefficient (epsilon: e) of your product then you apply the Beer Lambert Abs= e c l (l is the pathlength if you use cuvette of 1 cm then you can calculate c (concentration of product that appeared or substrate that disappeared) by Abs/el . Be careful with the units of e, to determine the C (usually in mM). If you have c in mM for instance and you are working in 1 mL you will know that you have let say if c = 0.2 mM 0.2 µMol in 1 mL . If now you know that you have a delta Abs in 1 min then means you have 0.2 µmol (200 nmol) per 1 min and you have to know how much enzyme you put in your cuvette (let say 2 nM) then your kcat (catalytic constant) will be 100 min-1. You can also work out activity as nmol/min/mg (then you need to know how much you put in the cuvette let say 1 µg in the 1 mL then meaning that you got 200 nmol/min for 100 µg so you mutliply by 10 to get 2000 nmol/min/mg or 2 µmol/min/mg that is also the enzyme activity.