I have NPA (nasopharyngeal aspirate) samples which have alkaline phosphatases that are interfering with our bioassay for the detection of muramyl-dipeptide (MDP). We want to denature the phosphatases and are considering to use heat or irradiation (so our volume is unaffected), however we need to keep the MDP in as perfect a state as possible for our bioassay. So how can I inactivate/denature the alkaline phosphatase with as little damage to MDP as possible?
have you thought about using an AP inhibitor such as vanadate? (http://www.jbc.org/content/259/6/3511.full.pdf). Depending on the pH and temperature/time I would not be surprised that the dipeptide could suffer some degradation with heating.
Good news! By heating at 65 degrees C for 30 minutes, the alkaline phosphatases show no more activity, but the MDP still maintains responsiveness under the NOD2 receptor. The responses didn't match up exactly to MDP that wasn't heat treated, but I think that's a pipetting issue more than heating.
We were avoiding inhibitors because we don't want to diluted our samples. Our assay isn't too sensitive saddly.
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