Hallo to everyone!
I have a question concerning the measurement of enzyme activity in vivo. Does anyone know a simple a reliable method to study the differential activity of enzymes in serum? Spectrometry and spectrophotometry suggestions are also appreciated ;)
Thank all in advance!
Cheers,
ms
please clearly mention the enzyme, and invivo meaning is it a animal study if so which animal
thanks
Ok, sorry...the enzyme is a phospholipase D and I would like to analyzed the activity in mouse and human sera....
Thanks Walter!
I have already checked there...there are a couple of methods quite old (reported in reviews 1997-1999); some of them, such as [3H]choline labeling, sounds really interesting for its high sensitivity. But I was wondering if someone is using a method routinely, which I can repeat easily.
Anyway, I will follow your suggestion, and I will go through carefully to find out the best solution. Then I will be back to discuss with all of you in detail!
Cheers!
HI Miguel!
Yes, I am allowed to do it...I think you have right. Since PL-D produces specifically PA (instead of PL-C) maybe I can focus my attemps on its measurement....
many thanks!!! I will come here through again for updates :)
Cheers!!
That is a comprehensive answear! Thanks!
My doubts have been arised since I am not sure if the PLD1 is working in serum as well as in the cells.
I had already identified this enzyme in mouse and human sera by MALDI MS and identified its glycosylated forms. That means that the assay I would have liked to carry out was an idea to understand if this enzyme works even outside the cell or if the PTMs have been just inactived it.
I am going to register to the webinar and I read right away that article.
Thank you again for the support and your kindness in prompt reply :)
Have a nice day,
Cheers
We have a group here working on phospholipases. I do remember that they use gas chromatography to follow product formation. That obviously avoids radioactivity in the assay. No idea what substrate they use though. There are standard methods to detect fatty acids with GC, typically an alkylating agent is added to modify the carboxylic acid group.
Thanks Kai, that is an option but unfortunately we have no GC in our Department...:(
You only need a cheap one, $7000 Euros should be more than enough to acquire one. But then again, you need training with it. One option would be to extract the fatty acids and send it to someone with a GC. I see you are in Hannover - I would check at the other university there.
I am registered!!
Many thanks, Miguel, I hope it can help me...;)
Have a nice day!!
hi,
I want to extract lipid from the mitochondria in the animal model. can anyone suggest me the procedure for lipid isolation from mitochondria.
Thanks in advance..
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