User:Paul Rothenberg/Notebook/Pauls Notebook/2014/06/02

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(Preparation of Apo-Mb)
Current revision (11:32, 4 June 2014) (view source)
(Preparation of Apo-Mb)
 
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==Preparation of Apo-Mb==
==Preparation of Apo-Mb==
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I am following the same procedure as [http://openwetware.org/wiki/User:Paul_Rothenberg/Notebook/Pauls_Notebook/2014/03/05| here] to prepare Apomyoglobin.
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I am following the same procedure as [http://openwetware.org/wiki/User:Paul_Rothenberg/Notebook/Pauls_Notebook/2014/03/05 here] to prepare Apomyoglobin.
The heme was extracted using the acid and 2-butanone method. Approximately 10 mL of the aqueous layer was produced. It was left to run dialysis overnight at 4°C in the 10 mM Na<sub>2</sub>CO<sub>3</sub> buffer. 500 mg of protein was used initially.
The heme was extracted using the acid and 2-butanone method. Approximately 10 mL of the aqueous layer was produced. It was left to run dialysis overnight at 4°C in the 10 mM Na<sub>2</sub>CO<sub>3</sub> buffer. 500 mg of protein was used initially.

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Preparation of Apo-Mb

I am following the same procedure as here to prepare Apomyoglobin.

The heme was extracted using the acid and 2-butanone method. Approximately 10 mL of the aqueous layer was produced. It was left to run dialysis overnight at 4°C in the 10 mM Na2CO3 buffer. 500 mg of protein was used initially.

Synthesis of Cobalt PPIX

Stoichiometric amounts of Cobalt Acetate (524 mg) were measured out, along with 500 mg of PPIX. 50 mL of DMF was set to reflux. Once refluxing, the cobalt acetate and PPIX were dissolved in minimum DMF and added via syringe to the refluxing DMF. It was let reflux for 30 minutes, and then chilled in an ice bath for 15 minutes. 500 mL of chilled water was added, and chilled for a further 10 minutes.

Once it was chilled, it was vacuum filtered. The mass of the filter paper before filtration was 0.0526 grams.


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