User:Melissa Novy/Notebook/CHEM-571/2012/10/03

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(Objectives)
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(Au/BSA Solutions for AA)
 
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==Objectives==
==Objectives==
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* Filter the cell protein solution made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/02|2012/10/02]] to obtain ADA using fast protein liquid chromatography (FPLC).
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* Use fast protein liquid chromatography (FPLC) on the cell protein solution made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/02|2012/10/02]] to obtain ADA.
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 18:17, 7 October 2012 (EDT)''':chromatography and filtering are both forms of separation but are not the same thing.
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* Make 10 mL of Au/BSA solutions at the usual mole ratios (60 through 170 Au:BSA) for atomic absorption. Please refer to the following table for more information.
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* Make 10 mL of Au/BSA solutions at the usual mole ratios for atomic absorption.
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 18:17, 7 October 2012 (EDT)''':"usual mole ratios" doesn't mean anything. be specific
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==Obtaining ADA with FPLC==
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 18:19, 7 October 2012 (EDT)''':where is the information about separating the ADA? how was it done? what conditions and parameter were important?  what buffers were used?
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* Binding and elution buffers made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/26|2012/09/26]] were obtained.
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* An AKTA Purifier and a 5 mL His nickel column were used to conduct FPLC.
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<br>
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Protocol
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*# Run about 100 mL of binding buffer through the AKTA system and column at a flow rate of 5 mL/min.
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*# Inject all of the cell protein solution into the system and set the flow rate to 10 mL/min.  Note that the composition of the solution is monitored with UV-vis at 280 nm.
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*# After all of the cell protein solution has been run through the 5 mL His nickel column, flush the system with about 200 mL elution buffer at a flow rate of 5 mL/min.
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*# Collect the ADA in elution buffer in glass test tubes and monitor the absorbance with UV-vis to determine the presence of ADA.
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*# Combine the solutions in the test tubes into one Falcon tube and store at 4°C until needed.
==Au/BSA Solutions for AA==
==Au/BSA Solutions for AA==
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* Due to a miscalculation, the stock solutions for HAuCl<sub>4</sub> and for BSA were combined.  The total number of moles of BSA and moles of HAuCl<sub>4</sub> in this new solution were calculated and then new volumes of BSA stock solution were determined:
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* Due to a miscalculation, the stock solutions for HAuCl<sub>4</sub> and for BSA were combined into 10 mL of mixed solution in water.  The total number of moles of BSA and moles of HAuCl<sub>4</sub> in this new solution were calculated and then new volumes of BSA stock solution were determined:
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*'''[[User:Abigail E. Miller|Abigail E. Miller]] 18:18, 7 October 2012 (EDT)''':give what you initially created to then combine. what was the original stock solution. what volumes of gold nad BSA were mixed before combining?
 
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Stock Solution Concentrations [M]'''
| align="center" style="background:#f0f0f0;"|'''Stock Solution Concentrations [M]'''

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Objectives

  • Use fast protein liquid chromatography (FPLC) on the cell protein solution made on 2012/10/02 to obtain ADA.
  • Make 10 mL of Au/BSA solutions at the usual mole ratios (60 through 170 Au:BSA) for atomic absorption. Please refer to the following table for more information.

Obtaining ADA with FPLC

  • Binding and elution buffers made on 2012/09/26 were obtained.
  • An AKTA Purifier and a 5 mL His nickel column were used to conduct FPLC.


Protocol

    1. Run about 100 mL of binding buffer through the AKTA system and column at a flow rate of 5 mL/min.
    2. Inject all of the cell protein solution into the system and set the flow rate to 10 mL/min. Note that the composition of the solution is monitored with UV-vis at 280 nm.
    3. After all of the cell protein solution has been run through the 5 mL His nickel column, flush the system with about 200 mL elution buffer at a flow rate of 5 mL/min.
    4. Collect the ADA in elution buffer in glass test tubes and monitor the absorbance with UV-vis to determine the presence of ADA.
    5. Combine the solutions in the test tubes into one Falcon tube and store at 4°C until needed.

Au/BSA Solutions for AA

  • Due to a miscalculation, the stock solutions for HAuCl4 and for BSA were combined into 10 mL of mixed solution in water. The total number of moles of BSA and moles of HAuCl4 in this new solution were calculated and then new volumes of BSA stock solution were determined:
Stock Solution Concentrations [M] '
[HAuCl4]0.026
[BSA]0.000003


  • There are 0.00000208 mol of HAuCl4 per 0.00008 L of solution.
  • There are 2.4E-10 mol of BSA per 0.00008 L of solution.
  • Please refer to the following table for calculations and concentrations:
Au:BSA Mol BSA Needed Mol BSA Added Volume BSA Added [L] Volume BSA Added [uL]
603.46667E-083.44267E-080.0022951112295.111111
800.0000000262.576E-080.0017173331717.333333
1002.08E-082.056E-080.0013706671370.666667
1201.73333E-081.70933E-080.0011395561139.555556
1281.625E-081.601E-080.0010673331067.333333
1300.0000000161.576E-080.0010506671050.666667
1321.57576E-081.55176E-080.0010345051034.505051
1331.56391E-081.53991E-080.0010266071026.606516
1341.55224E-081.52824E-080.0010188261018.825871
1361.52941E-081.50541E-080.0010036081003.607843
1381.50725E-081.48325E-080.000988831988.8309179
1401.48571E-081.46171E-080.000974476974.4761905
1600.0000000131.276E-080.000850667850.6666667
1701.22353E-081.19953E-080.000799686799.6862745


  • The protocol for making the AA Au/BSA solutions is as follows:
    1. Add 80 μL of Au/BSA stock solution to each test tube.
    2. Then add the given volumes of BSA stock solution to each test tube (refer to Volume BSA Added [μL]).
    3. Dilute all solutions to a final volume of 10 mL.
    4. Place the solutions in an 80°C oven for 4 hours.



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