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[ccp4bb] Offtopic: FAD enzymatic assay: a little bit more about my enzyme
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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Re: O/T: can a protein which dimerizes in solution crystallize as a monomer?
From: "Brown, Kate A" k {- dot -} brown {- at -} IMPERIAL {- dot -} AC {- dot -} UK
Date: 2008-12-02		Next message:
Subject: Re: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: conancao conan_cao {- at -} HOTMAIL {- dot -} COM
Date: 2008-12-02


Subject: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: michael nelson gnomenm {- at -} YAHOO {- dot -} COM
Date: 2008-12-02

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




CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
Previous message:
Subject: Re: O/T: can a protein which dimerizes in solution crystallize as a monomer?
From: "Brown, Kate A" k {- dot -} brown {- at -} IMPERIAL {- dot -} AC {- dot -} UK
Date: 2008-12-02		Next message:
Subject: Re: Offtopic: FAD enzymatic assay: a little bit more about my enzyme
From: conancao conan_cao {- at -} HOTMAIL {- dot -} COM
Date: 2008-12-02
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