I have been trying to isolate a P450 and measure activity using reduced Fe and Fe-CO spectral analysis to determine presence. I was unsuccessful for several weeks, but lately I have been getting peaks at 425nm. Does anyone know what this indicates? I am using LMG competent cells and purification by nickel columns
I am no expert in this field, but maybe these two publications could help you further:
http://www.jbc.org/content/277/27/24000.full
and
http://pubs.rsc.org/en/content/articlepdf/2007/np/b604190f
By the way, which spectrometer are you using for the characterization?
Thank you for your input, these articles gave me some very good ideas. I believe that the peak is the result of a degraded P450 due to harsh conditions of elution and wash buffers. After running a dialysis with a storage buffer immediately after eluting, the problem ceased to exist.
Could you upload tuhe Pic? Maybe that will be very useful for us to solve the problem.
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