endothelial lipase - concentration of the enzyme without loss of activity (Sep/04/2006 )
I was wondering if anyone has worked or is familiar with endothelial lipase. I am working with huvec cells stimulated with TNF-a to express the endothelial lipase (EL) and then treat with heparin thereby releasing EL in the media. The media is assayed for the enzyme activity using artificial substrate or natural substrate. However, the media generated is very weakly active and upon concentration using centricon loses more activity. Any idea how to concentrate this enzyme in an active form? Many thanks in advance for the any suggestion that you may have,
it could be that since you are concentrating your cell prep you are also concnetrating other possible substrates that would lead to reduced reactivity with the artificial substrate...
can you track protein amount by western blotting to show it is being broken down?
some other things to think about,,
are you carrying put all your purification steps on ice and with protease inhibitors?? i recommend you do both
after which you could try your centricon concentration, alternatively if you know the size of the protein you could remove some protein by spinning in size exclusion inserts..
hopw this helps
enjoy
can you track protein amount by western blotting to show it is being broken down?
some other things to think about,,
are you carrying put all your purification steps on ice and with protease inhibitors?? i recommend you do both
after which you could try your centricon concentration, alternatively if you know the size of the protein you could remove some protein by spinning in size exclusion inserts..
hopw this helps
enjoy
Jimmy thanks for the kind reply. The media that EL is secreted in is devoid of serum thus there is nothing in there but the secreted enzyme. Western confirms plenty of the intact enzyme. However the activity is low before and even lower before concentrating. The size of the intact enzyme is 68KD. What are the size exclusion inserts? I use Centricon30 which theoretically should retain the enzyme.
One possibility: lipase tends to be hydrophobic, may stick to the filter when you concentrate it. try to lyopholize your sample with a freez-drier.
Gehehunter, thanks for the suggestion. Don't you think lyopholization inactivates this enzyme?
One possibility: lipase tends to be hydrophobic, may stick to the filter when you concentrate it. try to lyopholize your sample with a freez-drier.
Gehehunter, thanks for the suggestion. Don't you think lyopholization inactivates this enzyme?
Well, this is a risk worth taking. Many enzymes tolerate this process well and I know some phospholipases (maybe not the one you are dealing with) are very hardy ones.