I have a problem with alkaline phosphatase detection in calcified VSMC using its chromogenic substrate, pNPP !!
I used Vascular smooth muscle cell line, I put them in Calcification media (in presence of CaCl2 (3mM) and beta-glycerophosphate (10mM) for 10 days), and in 10th day i did alkaline phosphatase test without commercial kit.
first i removed the medium and wash the cell mono-layes with PBS (2times), after this i lysed the cell using lysis buffer (0.1% triton in saline in presence of 2 protease inhibitor like PMSF, ...) and with help of blue tips i lysed the cells.
after that, i transferred the cells to microtubes and put them on ice in shaker, and every 5 min i vortex the microtubes, and finally centrifuge the cells in 15000rpm, 15min, 4C and with this supernatant performed ALP test
i added 50 micro-liter of supernatant to 100 micro liter alkaline buffer (containing Tris-HCl and glycine) and added 50 micro liter of pNPP (10-25mM) (which solved in alkaline buffer containing MgCl2 and ZnCl2) to them in dark condition.
But unfortunately the absorbance of positive (cells in calcification media) and negative control (cells in normal media) didn't have significant difference!!!!
Could you kindly guide me about these? and tell me what is the problem?
Thanks a lot
Best regards
Fatemeh
before you do the analysis treat with 5% H2O2 for 30 min in methanol to reduce the endogenous activity of PA
Thanks dear Eliseo for your response. H2O2 dose not have destroying effects on alkaline phosphatase or dose not interference in enzymatic reaction of ALP? PMSF and leupeptin in concentration of 1mM cant inhibit protease activity?
Thank you very much
Is it possible, that your cells just contain AP regardless of the treatment?
The inhibitor of AP is levamisol (commercially available).
I would try to inhibit AP in the negative control to get a AP-independent result. If negative and specimen look still the same, there must be some unspecific binding or reaction with the other reagens.
Is it possible to do a washing step after the developement?
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