Hello all,
I am over-expressing acid sphingomyelinase enzyme in HeLa cells. Theenzyme shows expression on western blot but I do not have any activity in in vitro assays. I have checked the activity through Mass Spec as well as radioactivity. Please suggest where could I be going wrong? Thanks!
Hi there,
If your enzyme of interest is produced then there might be an issue with its folding/stability (if not properly folded/matured the protein will exhibit no activity), cell extraction (possibly provoking the loss of enzyme activity) and/or with the assay itself (compatibility with the source of enzyme you use).
This could be happened by the misfolded enzyme or by an inaccurate activity assay protocol...
Which enzyme activity assay you used (in-house developed, commercial?)
(hydrolysis/incubation conditions, buffers, substrates, conc, volumes, detection technique, etc..)
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