Typically when I do in vitro osteogenic differentiation, I perform a protein assay as well on the cell lysate upon harvesting, and normalize the ALP activity to protein concentration.
However, I'm wondering if this method is a reliable measure of ALP activity per unit, especially if I have a scenario whereby I'm adding, say, rhBMP2 to one group's mineralizing medium, and have a negative control group that just has mineralizing medium. Wouldn't the higher intracellular BMP2 reflect a higher protein concentration that doesn't necessarily mean a higher cell count? Is there a way to normalize ALP activity per cell or comparable unit, other than counting the cells when harvesting?
Maryanne Achieng
Sebastian Schmitt
thank you for your answer! That actually makes sense, since BMP2 is an extracellular signaling molecule. My doubts lay in an experiment gone awry, where my negative control exhibited significantly more normalized ALP activity than the BMP groups; trying to troubleshoot at this point. I might go with a Western blot to determine the amount of ALP in the lysate. I got about 2.4 mg/ml of protein for the BMP group and 1.1 for control. I was working with 500 ul of lysate for each group. I typically add 100 ng BMP
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