User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03

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(Procedure)
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==Procedure==
==Procedure==
* Column from AKTA purifier were washed with 100mL binding buffer at pH 7.5 with Tris, NaCl, and 30mM of imidazol.  
* Column from AKTA purifier were washed with 100mL binding buffer at pH 7.5 with Tris, NaCl, and 30mM of imidazol.  
 +
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 17:56, 7 October 2012 (EDT)''':what are the tris nad NaCl concnetrations? what is AKTA purifier?
* Protein supernatant filtered from yesterday was injected into column.
* Protein supernatant filtered from yesterday was injected into column.
* Protein supernatant were run through the column with AKTA purifier. Flow through were collected in 50ml falcon tube just in case protein failed to bind to column.
* Protein supernatant were run through the column with AKTA purifier. Flow through were collected in 50ml falcon tube just in case protein failed to bind to column.
Line 17: Line 18:
* UV-vis showed concentration protein elution at 280nm absorptivity, and indicated the elution test tube with most concentrated ADA protein.
* UV-vis showed concentration protein elution at 280nm absorptivity, and indicated the elution test tube with most concentrated ADA protein.
* Two test tubes of ADA protein were collected in a 15mL falcon tube, and flow through of the protein are stored in 4C.
* Two test tubes of ADA protein were collected in a 15mL falcon tube, and flow through of the protein are stored in 4C.
 +
 +
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 17:56, 7 October 2012 (EDT)''':more information - flow rates? time? any specific parameters for the FPLC.
==Discussion==
==Discussion==

Revision as of 17:56, 7 October 2012

Experimental Biological Chemistry I Main project page
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Purpose

  • Run protein supernatant through nickel-filled column in order to separate adenosine deaminase from the rest of protein in supernatant.
  • Remake Au/BSA solutions with different Gold to BSA ratios for experiments for next week.

Procedure

  • Column from AKTA purifier were washed with 100mL binding buffer at pH 7.5 with Tris, NaCl, and 30mM of imidazol.
  • Abigail E. Miller 17:56, 7 October 2012 (EDT):what are the tris nad NaCl concnetrations? what is AKTA purifier?
  • Protein supernatant filtered from yesterday was injected into column.
  • Protein supernatant were run through the column with AKTA purifier. Flow through were collected in 50ml falcon tube just in case protein failed to bind to column.
  • 200mL elution buffer were run through the column while collecting the binded ADA protein in separate test tubes.
  • UV-vis showed concentration protein elution at 280nm absorptivity, and indicated the elution test tube with most concentrated ADA protein.
  • Two test tubes of ADA protein were collected in a 15mL falcon tube, and flow through of the protein are stored in 4C.
  • Abigail E. Miller 17:56, 7 October 2012 (EDT):more information - flow rates? time? any specific parameters for the FPLC.

Discussion

  • Protein supernatant were run through the column. The nickel inside the column binds to the histidine head of Adenosine Deaminase protein. Adenosine Deaminase is the only protein among protein supernatant with histidine head, thus ADA protein should be the only protein sticking to the nickel column.
  • Once elution buffer were run, the high imidazol concentration within elution buffer is subject to competitive binding with the nickel in the column, allowing the nickel to release adenosine deaminase.
  • ADA in response is released from the column and collected into elution test tubes.



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