I´m about to set up an off-target assay for a PLA2 enzyme and I´m wondering if i do always have to dispense my substrates in liposomes when doing a QRFRET assay
I don't have personal experience with this situation, but it seems to me that the assay could work using a non-ionic detergent such as Triton X-100 to dissolve the fluorescent lipid substrate, instead of using liposomes. A TX-100 concentration of at least 0.013% would be necessary. This is the critical micellar concentration. I have done a FRET assay using lipids dissolved in this detergent where the final TX-100 concentration was 0.04% (final).
Hi Gernot, a loooong time ago I was doing enzymatic assays of PLA2's isolated from snake venom.
I was using an automatic pH titrator and added PLA2's to a liposome mixture. The assay took a long time, but it gave me results.
I experimented with soluble PLA2 substrates, but never got good results.
Since PLA2's need to attach to phospholipid surfaces in order to do their job, I think that you have to use liposomes to measure their activity, unless someone has come up with a better way of measuring these guys.
Hi Gernot
We have several interesting off-target assays, both for sera and cells, see attached paper for example off cell-based assays.
Feel free to contact me if you want more information or have questions.
Best regards
Teodor
Article Mapping the immunogenicity of therapeutic protein products
@Teodor. First of all, a full text of your article is not available on ReseachGate and secondly, there is no mention of PLA2 enzymes in your abstract.
Do you care to elaborate?
First of all, thank´s for your input.I´ve been out of the office and I´ll be back to you in due time
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