I am having a problem when assaying ATP citrate lyase from the crude extract of Y. lipolytica (continuous assay) that is the exogenous activity is too high. Based on literature, I suspect it is due to activity of complex I respiratory chain (NADH-CoQ oxidoreductase).
Hence, I am planning to partial purify ATP citrate lyase by fractionally precipitate with ammonium sulphate it out from the crude extract. Can I know the % of ammonium sulphate suitable for this precipitation that it does not include complex I in the precipitate?
Thanks in advance.
Is there an inhibitor of complex I respiratory chain that you can get that does not inhibit ATP citrate lyase? You could add it into the assay to remove the undesired background activity.
Is there an inhibitor of complex I respiratory chain that you can get that does not inhibit ATP citrate lyase? You could add it into the assay to remove the undesired background activity.
I'm afraid you'll have to try empirically, but I'm rather pessimistic that this will work. Precipitation with ammonium sulphate has very low resolution. I'd recommend rather some chromatography. Or the inhibitor as suggested by Adam.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays