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Protein expression - (Oct/15/2015 )

Hi,
I doing protein expression p450 in BEVS. I got positive result on western blot analysis. However in my spectral quamtification, i dont get the peak of wavelenght at 450, i just get at 420. Any idea to solve this problem? Here my method

1. The expressed protein pellet were resuspended with reaction buffer (01M PBS pH7+20% glycerol+1mM EDTA+0.5% sodium cholate+0.4% triton N-101)
2. 1mg Sodium dithionate was added to Rwaction buffer
3. The buffer was aliqout into 2 cuvette, and run for measuring the baseline
4. One cuvette was taken out for introducing to CO (bubbling around 40)
5. Re-measure the sample for several time until the peak of 450 was obtained.
(in this step, even after an hour i still dont get the 450 peak)

-JohnE-

I've read that P450 needs to be reduced and in complex with carbon monoxide to express a 450 nm peak.

 

What are each of the components in your reaction buffer purpose?

What is the purpose of sodium dithionite?

Is there a reducing agent (like DTT) in your reaction buffer?

Oh, I see you are bubbling CO. Around 40 what? Temperature or duration? Maybe you're not bubbling long enough?

-Michael Starr-