I need to know if an enzyme I added is working by spectometry of Malonyl-CoA, but I do not know how to purify it, does anyone?
Hi there,
Malonyl-CoA is commercially available from Sigma for instance. Expensive but if you don't need a lot of it...
Hello!
I'm making a new pathway in S. cerevisiae for overexpressing fatty acids. This pathways consist in several enzymes, including Acetyl-CoA Carboxylase that will produce Malonyl-CoA.
Now I need to detect the metabolits from the pathway, to see if the enzyme is working, and, quantitatively, verify the difference between the non tranformed and the transformed one.
I am accepting suggestions :)
ok, I see. What you need is not a quantification method for mal-CoA but rather an activity assay for acetyl-CoA carboxylase.
My idea is to use an assay developed in the lab of Georg Fuchs in Freiburg/Germany. There is a paper that describes the use of this mal-CoA reductase to measure the activity of ac-CoA carboxylase. They do have an expression plasmid for the gene that can be used to produce the enzyme in E. coli. A partially purified preparation (e.g. after heat denaturation of E. coli proteins and centrifugation) will do for most experiments. The only important thing is that the endogenous E. coli enzyme is removed. However, there are several other assays that can be used. Your choice depends on the equipment you have at hands in your lab.
Best wishes
Jürgen
It's cheaper from CoALA Biosciences than from Sigma. They also have an NMR spectrum on the product page for malonyl-CoA if that helps with your research.
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