I am doing the neutral endopeptidase (NEP) activity assay in the pancreas and lungs by using fluorosent substrate (Mca-RPPGFSAFK(Dnp)-OH Fluorogenic Peptide Substrate) and NEP Specific inhibitor (thiorphan). In case of lungs, the inhibitor is inhibiting the total fluorescent as compare to control but in pancreas there is a little inhibition or almost no difference between the treatment and control group. The reason may be the activation of the other protease enzymes in the pancreas that cleaves the fluorescent substrate as compare to lungs.
Can anyone suggest the protease inhibitor effectively inhibit the activated proteases in pancreas without affecting the NEP activity? Otherwise, what is the better possible solution to prevent non-specific hydrolysis of the fluorescent substrate in pancreas homogenate?
According to Uniprot NEP is zinc dependent peptidase so you cannot use metal chelators like EDTA, EGTA or phenantroline. Try using PMSF, aprotonin, leupeptin or pepstatin. With this mix you can inactivate most of the proteases present in your lysates. Pancreas produce digestive enzymes so it is highly propable that PMSF and aprotonin will work. Be sure to have all the inhibitors fresh. PMSF for example is very unstable in water solutions.
Hi David,
Thank you for your valuable answer. I used the PMSF Aprotenin, leupeptin and Pepstatin while doing homogenisation process but not while doing the assay. I did the homogenisation last month, it is not fresh homogenate. So should i use again these protease inhibitors while doing the assay.
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