I'm working on enzyme activity of SufS for Iron-Sulfur Cluster. I have a problem of positive control, while testing my gene (Enzyme) that show no activity, the procedure is follow.
Positive Control: L-Alanine (100mM) 10 microL, KPP buffer (10 mM K2HPO4 and KH2PO4) 890 microL Incubate on 30C for 30 min, on 100C for 10 min, centrifuge @12000rpm for 10 min.
Then take 900 microL plus 100 microL NAD (100mM) incubate in the UV-2450 Spectroscopy for 5 min then add 10 microL ADH (L-alanine dehydrogenase) Dilute into KPP buffer with 9:1, but show no activity and suppose to be show high activity.
Alanine as substrat here.
Is this enzyme inhibited by oxygen? Many FeS enzymes do not work in the presence of oxygen.
Hi there,
I have just trivial issues:
is the spec set at the right wavelength to follow NAD reduction?
ADH is also alcohol dehydrogenase so confusion is possible... Also you are using L-Ala in your control so you have to use L-Ala dehydrogenase.
Last trivial issue: your enzyme might have simply lost its activity...
@Dominique Liger
1) your first issue might be right, i'll check it out. what you suggest?
2) Yeah! i used L-ADH
3) This enzyme is just open three day before, and have in safe hand.
Hi again,
About the spec settings, you have to set the wavelength to 340nm and check it working properly (with NADH sample for instance).
Also run the classical assay for L-ADH as described by the manufacturer if not already done to actually check the activity of the enzyme. The fact that the sample is recent doesn't prove it is active automatically in any assay.
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