User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/10/03

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''' Purifying Protein via FPLC'''
''' Purifying Protein via FPLC'''
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* The FPLC column was washed with 100mL binding buffer (pH 7.5 with Tris, NaCl, and 30mM of imidazol).
+
* The FPLC column was washed with 100mL binding buffer (pH 7.5 with 0.03M Imidazole, 0.02M Tris, and 0.5M NaCl).
-
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 20:14, 7 October 2012 (EDT)''':what are the conc. of hte tris and NaCl.  
+
* The filtered protein supernatant from yesterday was injected into the column.
* The filtered protein supernatant from yesterday was injected into the column.
* The flow through from the column was collected in the same 50ml falcon tube that previously contained the protein supernatant as a precaution in case protein failed to bind to column.
* The flow through from the column was collected in the same 50ml falcon tube that previously contained the protein supernatant as a precaution in case protein failed to bind to column.
* 200mL elution buffer was pushed through the column while collecting the binded ADA protein in 5mL aliquots in separate test tubes.  
* 200mL elution buffer was pushed through the column while collecting the binded ADA protein in 5mL aliquots in separate test tubes.  
-
**NOTE: The nickel column binds to the histidine complex of Adenosine Deaminase (ADA) protein. Adenosine Deaminase is the only protein among protein supernatant with histidine complex. So theoretically, the ADA protein will be the only protein that sticks to the nickel column. The elusion buffer contains a high concentration of imidazole that competes with the nickel to bind to the histidine of the ADA protein. Theoretically, the ADA protein will be more attracted to the imidazole in the elusion buffer than the nickel in the column. Therefore, the ADA protein would flow through the column with the elusion buffer.
+
**NOTE: The nickel column binds to the histidine complex of Adenosine Deaminase (ADA) protein. Adenosine Deaminase is the only protein among protein supernatant with histidine complex. So theoretically, the histidine tag on the ADA protein will bind to the nickel column. The elusion buffer contains a high concentration of imidazole that competes with the nickel to bind to the histidine of the ADA protein. Theoretically, the imidazole will replace the histag, binding to the nickel in the column, such that the the ADA protein and histag would flow through and off the column with the elusion buffer.
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 20:14, 7 October 2012 (EDT)''':no the ADA protein is not attracted to the imidizole, the histidine tag on the ADA binds the nicklel. the imidizole replaces the histag in binding to the nickel so that the ADA with the histag is free to flow off hte column.
+
* The AKTP purifier (that runs the FPLC) took real-time UV-vis at 280nm of the run through and elutent from the column. The 280nm peak indicates the presence of protein in the elutent. This UV-vis made it be feasible to identify the test tubes containing the ADA protein.  
* The AKTP purifier (that runs the FPLC) took real-time UV-vis at 280nm of the run through and elutent from the column. The 280nm peak indicates the presence of protein in the elutent. This UV-vis made it be feasible to identify the test tubes containing the ADA protein.  
* The ADA protein was collected in two 5mL test tubes and combined into a  15mL falcon tube. The falcon tubes were stored at 4°C.
* The ADA protein was collected in two 5mL test tubes and combined into a  15mL falcon tube. The falcon tubes were stored at 4°C.
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'''Preparing Au/BSA Solutions of AA'''
'''Preparing Au/BSA Solutions of AA'''
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*The stock solutions for HAuCl<sub>4</sub> and BSA were combined. The moles of each component were calculated in this "new stock" and the BSA needed was calculated so that the desired Au/BSA ratios could be obtained. Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03| Melissa's entry]] for tables and specifics including moles, volumes, and edited protocol.  
+
*Because of a miscalculation, the stock solutions for HAuCl<sub>4</sub> and BSA were combined to create a 10mL mixed HAuCl<sub>4</sub> and BSA aqueous solution. The moles of each component were calculated in this "new stock."
-
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 20:14, 7 October 2012 (EDT)''':you need more details what stock did you start with? what ratios did you end up with? what did you do with the samples? you can reference the caluclations in Melissa's notebook but you need to explain what you are doing.  
+
**[HAuCl<sub>4</sub>]: 0.026M
 +
**[BSA]: 0.000003M
 +
*0.00008L (0.00000208 mol HAuCl<sub>4</sub> and 2.4E-10 mol BSA) of "new stock" solution was added to each solution. 
 +
*The BSA needed was calculated so that the desired Au/BSA ratios could be obtained. Please refer to [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03| Melissa's entry]] for tables and specifics including moles and volumes of BSA added.
 +
*The solutions were diluted to a final volume of 10mL, capped, covered in aluminum foil, and placed in a 80°C oven for 4 hours.
==Data==
==Data==

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Objective

  • to purify filtered protein through FPLC and
  • to remake Au/BSA solutions to run through Atomic Absorption.

Description

Purifying Protein via FPLC

  • The FPLC column was washed with 100mL binding buffer (pH 7.5 with 0.03M Imidazole, 0.02M Tris, and 0.5M NaCl).
  • The filtered protein supernatant from yesterday was injected into the column.
  • The flow through from the column was collected in the same 50ml falcon tube that previously contained the protein supernatant as a precaution in case protein failed to bind to column.
  • 200mL elution buffer was pushed through the column while collecting the binded ADA protein in 5mL aliquots in separate test tubes.
    • NOTE: The nickel column binds to the histidine complex of Adenosine Deaminase (ADA) protein. Adenosine Deaminase is the only protein among protein supernatant with histidine complex. So theoretically, the histidine tag on the ADA protein will bind to the nickel column. The elusion buffer contains a high concentration of imidazole that competes with the nickel to bind to the histidine of the ADA protein. Theoretically, the imidazole will replace the histag, binding to the nickel in the column, such that the the ADA protein and histag would flow through and off the column with the elusion buffer.
  • The AKTP purifier (that runs the FPLC) took real-time UV-vis at 280nm of the run through and elutent from the column. The 280nm peak indicates the presence of protein in the elutent. This UV-vis made it be feasible to identify the test tubes containing the ADA protein.
  • The ADA protein was collected in two 5mL test tubes and combined into a 15mL falcon tube. The falcon tubes were stored at 4°C.


Preparing Au/BSA Solutions of AA

  • Because of a miscalculation, the stock solutions for HAuCl4 and BSA were combined to create a 10mL mixed HAuCl4 and BSA aqueous solution. The moles of each component were calculated in this "new stock."
    • [HAuCl4]: 0.026M
    • [BSA]: 0.000003M
  • 0.00008L (0.00000208 mol HAuCl4 and 2.4E-10 mol BSA) of "new stock" solution was added to each solution.
  • The BSA needed was calculated so that the desired Au/BSA ratios could be obtained. Please refer to Melissa's entry for tables and specifics including moles and volumes of BSA added.
  • The solutions were diluted to a final volume of 10mL, capped, covered in aluminum foil, and placed in a 80°C oven for 4 hours.

Data

Image:ADA_HisTrap_FPLC.PNG The UV-vis at 280nm of the elutent from the nickel column.


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