User:Melissa Novy/Notebook/CHEM-571/2012/10/02

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(Autocreate 2012/10/02 Entry for User:Melissa_Novy/Notebook/CHEM-571)
Current revision (16:14, 26 November 2012) (view source)
 
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==Entry title==
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==Objectives==
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* Insert content here...
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* Obtain proteins from cells prepared on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/26|2012/09/26]].
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** Adenosine deaminase (ADA) will be separated from the rest of the cell proteins on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03|2012/10/03]].
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==Protein Purification==
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* The cells were lysed using a Misonix XL-2000 sonicator to allow direct manipulation of organelles, proteins, and other cell components in solution.
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* The sonicated cell solution was centrifuged at 4°C and 18,000 rpm for a period of 2 hours.
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** Note that cooling the solutions prevents cell proteins from denaturing.
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** The resultant supernatant was then filtered with a 450 nm nylon filter to remove all large cell components.  The filtrate is expected to contain cell proteins.
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* The filtrate containing cell proteins, including ADA, was refrigerated at 4°C until needed.
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* Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/10/02|Dhea Patel's entry]] for the detailed protocol for protein purification and proper disposal of biowaste.
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Objectives

  • Obtain proteins from cells prepared on 2012/09/26.
    • Adenosine deaminase (ADA) will be separated from the rest of the cell proteins on 2012/10/03.

Protein Purification

  • The cells were lysed using a Misonix XL-2000 sonicator to allow direct manipulation of organelles, proteins, and other cell components in solution.
  • The sonicated cell solution was centrifuged at 4°C and 18,000 rpm for a period of 2 hours.
    • Note that cooling the solutions prevents cell proteins from denaturing.
    • The resultant supernatant was then filtered with a 450 nm nylon filter to remove all large cell components. The filtrate is expected to contain cell proteins.
  • The filtrate containing cell proteins, including ADA, was refrigerated at 4°C until needed.
  • Please refer to Dhea Patel's entry for the detailed protocol for protein purification and proper disposal of biowaste.


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