I need to determine ALP activity in osteoblasts. I want to do it by colorimetric method but the reaction does not seem to work, even after incubating the plate at 37 for 1 hour I am not getting yellow colored product. I will be grateful if somebody could give a working buffer composition.
Please refer to the protocol from Sigma Aldrich regarding buffer composition
http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-acid-phosphatase.html
To analyse ALP activity of Osteblasts you will need two different buffers.
ALP-Lysisbuffer (to extract the ALP from the cells); 80 - 100µl/ 24-well
1,5M TRIS (pH10)
1mM ZnCl2
1mM MgCL2
1% Triton X-100
ad 500 ml H2O
addition of Aprotinin and PMSF directly before use is recommended
ALP-Buffer
0,1M DEA (pH 9,8)
0,1 % Triton X-100
ad 500 ml H2O
pNPP
3,6 mM
ad ALP-Buffer
10µl lysat + 100 µl pNPP
incubation time: 30min, 37°C
reaction stop: 100 mM NaOH (yellow color)
With kind regards
Anne-Helen Lutter
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