caspase activity assay - don't understand (Jun/10/2007 )
Hi all,
I just started in a new lab, and I've been given the task of doing a caspase activity assay, but I don't understand the rationale behind it. I've the protocol, which needs me to add a reaction buffer ( HEPES, EDTA, DTT, KCl and MgCl2) that is supplemented with protease inhibitors and caspase substrate ( caspase 3 substrate : Ac-DEVD-AFC and caspase 9 substrate: Ac-LEHD-AFC) Then I've to incubate it and measure fluorescence. I'd greatly appreciate some help in understanding why I'm doing this.
Haven't been able to find much on pubmed books and google.
Thanks for your help!
not sure if its relevant but we look for caspase (in tissue ) to check for apoptosis (rather than necrosis or cell damage)
dom
I just started in a new lab, and I've been given the task of doing a caspase activity assay, but I don't understand the rationale behind it. I've the protocol, which needs me to add a reaction buffer ( HEPES, EDTA, DTT, KCl and MgCl2) that is supplemented with protease inhibitors and caspase substrate ( caspase 3 substrate : Ac-DEVD-AFC and caspase 9 substrate: Ac-LEHD-AFC) Then I've to incubate it and measure fluorescence. I'd greatly appreciate some help in understanding why I'm doing this.
Haven't been able to find much on pubmed books and google.
Thanks for your help!
activated caspases cleave their substrates - here in your kit artificial substrates tagged with fluorochromes to monitor by change in fluorescence-
protease inhibitors should inhibit non-caspase proteases; other components give an optimum buffered medium for measuring caspase activity; you analyze caspase-dependence of apoptosis
is there a control with caspase inhibitors included?
Hmm my whole experiment is about inhibiting apoptosis... i think so. Actually I don't really know what's going on. Will have to read up more i guess. I just started college not too long ago. Was thrown into this as part of a programme. Thanks!
the term for those assay you perform is "apoptosis assay"; it doesn´t matter if you look for apoptotic or anti-apoptotic effects
there is a nice book about apoptosis to download at Roche homepage
ooh ok thanks!
This is my first post and a want to say a big hello! I'm very happy that i found this forum!
Now,on topic, could you please post the link to this apoptosis book at roche homepage?
Thanks in advance 
Well,the links @roche were bad,so to whom may be concerned there is an alternate location
manual_apoptosis.pdf -ROCHE-
Cheers!
hai anyone know which is the best apoptosis assay to choose for .? because there are so many in the market . thanks for ur helpapoptosis