I want to do analyses on microalgal alkaline phosphatase activity (APA), using the hydrolysis of p-NPP substrate to p-NP. I have para-nitrophenyl phosphate as 5 mg tablets (https://www.sigmaaldrich.com/catalog/product/mm/20106).
The suggested buffers for p-NPP substrate preparation are 0.1 M glycine buffer containing 1 mM MgCl2 and 1 mM ZnCl2, pH 10.4, or 1 M diethanolamine buffer containing 0.5 mM MgCl2, pH 9.8. However, I want to know if there are any alternatives to these buffers. For instance, Tris-HCl buffer.
You can use any buffer you want to use. Choose a buffer that is well-suited to the pH you want to run the reaction in. For Tris, the pKa is 8.1 at 25oC, so it is best-suited for pH 7.6-8.6. The buffers you mentioned seem to be intended for reactions at pH about 10, which is too high for Tris. The enzyme activity may be affected by both the pH and the buffer.
Greetings. Since your substrate get oxidised at alkaline pH you are looking for alternative buffer. If so, you keep proper control in double beam UV.VIS Spectrophotometer and perform the experiment. You may get meaningful results.
Adam B Shapiro Thank you for your answer! I have another question on this same topic. I posted it here: https://www.researchgate.net/post/Alkaline_phosphatase_activity_test-concentrations_volumes_and_incubation_period_ratio
Could you please provide any tips on that, if possible?
Hi Aigars, you can try out the standard buffer 100 mM Tris-HCl, pH 9.5 , 100 mM NaCl , 5 mM MgCl2. Thanks.
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