Mike:
It seems FAD is readily dissociable for your protein.Then, trust me, you
got to do it anaerobically in order to see the 450nm decrease upon reduction of
the enzyme by the substrate(better use excess substrate).
Sincerely,
Hongnan Cao
UCR
Date: Tue, 2 Dec 2008 11:11:53 -0800From: [EMAIL PROTECTED]: [ccp4bb] Offtopic:
FAD enzymatic assay: a little bit more about my enzymeTo: CCP4BB@JISCMAIL.AC.UK
Dear all,Thank you for all your kind replies.Here is a little bit more about
the enzyme and how I carry out the assay at the first place.My enzyme is a
lipid desaturase, originally from plant but overexpressed in bacteria. FAD
serves as a co-factor for this enzyme, in which FAD is reduced to FADH2. My
goal is set up an assay that would allows me to continuously monitor the
progress of the reaction. And I didn't want to use HPLC to analyze the final
product since that would take a lot time and we don't have an instrument
readily available to us. I wish FAD could be an alternative way since FAD will
have different Abs in reduced or oxidized forms. I set up assay is a regular
lab setting (not anaerobic), add FAD, substrate, ions and incubate. I finally
add enzyme to initialize the reaction. I expect to see some decrease of the Ab
at 450 nM. But I didn't.I have several concerns, one is the autooxidisability
of FAD, how fast FADH2 would be reoxidized by O2 in the air or by the O2
dissolved in solution. The second cocern is how fast the FAD reaction will
go.Please advise.Thank you!Mike
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