User:Melissa Novy/Notebook/CHEM-571/2012/11/14

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** The molar absorptivity coefficient of HRP at 403 nm is 1.02×10<sup>5</sup> M<sup>-1</sup> cm<sup>-1</sup>.
** The molar absorptivity coefficient of HRP at 403 nm is 1.02×10<sup>5</sup> M<sup>-1</sup> cm<sup>-1</sup>.
* Dialyze fractions of ADA made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06|2012/11/06]].
* Dialyze fractions of ADA made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/03|2012/10/03]] and [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06|2012/11/06]].
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** Please refer to [[User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I/2012/11/14|Keyun Wang's entry]] for the dialysis procedure, lengths of dialysis tubing used for each fraction of ADA, and notes and observations.
==Au-ADA Solutions==
==Au-ADA Solutions==
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==ADA Activity Assay with Adenosine==
==ADA Activity Assay with Adenosine==
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* Frac 1,3 of ADA, made on x, was used.  Its concentration was 10.35 μM, as determined by Bradford assay.
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* Frac 1,3 of ADA, made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06|2012/11/06]], was used.  Its concentration was 10.35 μM, as determined by Bradford assay.
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* It was diluted to 1 ng/μL, then to 0.1 ng/μL using phosphate buffer, prepared on x.
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* It was diluted to 1 ng/μL, then to 0.1 ng/μL using phosphate buffer, prepared on [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/13|2012/11/13]].
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** Please refer to [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/11/13|Puja Mody's entry]] for phosphate buffer calculations and actual concentrations.
   '''Initial concentration of ADA: 10.35 μM in 1 mL of solution'''
   '''Initial concentration of ADA: 10.35 μM in 1 mL of solution'''
   '''(10.35×10<sup>-6</sup>)(0.001 mL) = 1.035×10<sup>-8</sup> mol ADA'''
   '''(10.35×10<sup>-6</sup>)(0.001 mL) = 1.035×10<sup>-8</sup> mol ADA'''

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Objectives

  • Make Au-ADA solutions at mole ratios of Au:ADA from 60 to 150.
  • Make Au-HRP solutions at mole ratios of Au:HRP from 60 to 150.
    • Analyze the concentration of HRP stock solution with UV-vis.
    • The molar absorptivity coefficient of HRP at 403 nm is 1.02×105 M-1 cm-1.
  • Dialyze fractions of ADA made on 2012/10/03 and 2012/11/06.
    • Please refer to Keyun Wang's entry for the dialysis procedure, lengths of dialysis tubing used for each fraction of ADA, and notes and observations.

Au-ADA Solutions

  • Ten solutions at Au-ADA mole ratios of 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 were made.
  • Please refer to Dhea Patel's entry for theoretical and actual calculations of stock solutions, and volumes of stock solutions and deionized H2O added to each solution.
  • Note that ADA made on 2012/10/03 was used to make the solutions. The solution of HAuCl4 was made on 2012/09/05.

Au-HRP Solutions

  • Ten solutions at Au-HRP mole ratios of 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 were made.
  • Please refer to Dhea Patel's entry for theoretical and actual calculations of stock solutions, and volumes of stock solutions and deionized H2O added to each solution.
  • Note that 130, 140, 150 Au-HRP solutions were recalculated to account for a new stock solution at different concentration of HRP.
    • The concentration of the new HRP stock solution was calculated from its absorbance at 403 nm and its molar absorptivity coefficient.
    • Its concentration was calculated to be 28.5 μM.
  Abs.: 2.1
  Path length (b): 1 cm
  Molar absorptivity coefficient (ε) = 1.02×105 M-1 cm-1
  Concentration (c) = 2.1/(1 cm)(1.02×105 M-1 cm-1) = 28.5 μM

ADA Activity Assay with Adenosine

  • Frac 1,3 of ADA, made on 2012/11/06, was used. Its concentration was 10.35 μM, as determined by Bradford assay.
  • It was diluted to 1 ng/μL, then to 0.1 ng/μL using phosphate buffer, prepared on 2012/11/13.
    • Please refer to Puja Mody's entry for phosphate buffer calculations and actual concentrations.
  Initial concentration of ADA: 10.35 μM in 1 mL of solution
  (10.35×10-6)(0.001 mL) = 1.035×10-8 mol ADA
  (1.035×10-8 mol)(42000 g/mol) = 0.0004347 g ADA/mL
  (0.0004347 g ADA/mL)(1 mL/1000 μL)(109 ng/1 g) = 434.7 ng/μL
  (434.7 ng/μL)(V1) = (1 ng/μL)(1000 μL)
  V1 = 2.3 μL
  (1 ng/μL)(V1) = (0.1 ng/μL)(1000 μL)
  V1 = 100 μL

References

R&D Systems ADA Activity Assay
AU Biomaterials Design Lab ADA Activity Assay



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