I'm doing ALP assays on C2C12 myoblast as an osteogenic marker on the change of the differentiation pathway. But my negative control (C2C12 without osteogenic factors) gave me high values of ALP, and similar to the samples.
I've tested C2C12 cultured in DMEM + different FBS content (10%-0%) without any osteogenic factors added and they still gave me ALP activity of 1.48-1.55 U/ml which I believe it shouldn't.
I hope to have some explanations and ideas on this. Thank you.
Unspecific phosphatases are fairly common in biological fluids. Is the phosphatase activity you get linearly dependent on serum concentration? Are you sure that non-activated cells have no phosphatase, rather than just a lower background?
Engelbert Buxbaum The phosphatase level I got were not linearly dependent on serum concentration. They just gave me around the same values mentioned above. Some suggest me to correct the values to zero by using the blank. As it can be seen that they gave background readings. What do you think?
Ok, if the phosphatase activity does not increase in line with serum concentration, than serum is unlikely to be the source. I'd still try to establish where that activity comes from, and add that to my discussion. Reviewers have the habit of asking such things ;-)
The activity in activated cells should be significantly higher than in the untreated cells. You need a standard deviation for the values of both, then you can do a t-test for the H0: both are identical. Only if you can reject H0 is it worth to proceed.
One last thing: Enzyme activities should no longer be reported as units (U = µmol/min), but as katal (kat = mol/s), 1 U = 16.7 nkat.
Engelbert Buxbaum thanks for your suggestions there. I will try them. :)
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