User:Melissa Novy/Notebook/CHEM-571/2012/10/23

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(HRP-Luminol Assay)
Current revision (17:34, 26 November 2012) (view source)
 
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   '''New concentration of sodium carbonate: (1 × 10<sup>4</sup> mol)×(0.120 L) = 0.833 mM'''
   '''New concentration of sodium carbonate: (1 × 10<sup>4</sup> mol)×(0.120 L) = 0.833 mM'''
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==Au-Lysozyme Solutions==
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* The protocol was based on Lee and Geckeler's paper, [http://onlinelibrary.wiley.com/doi/10.1002/jbm.a.34020/abstract Cytotoxicity and cellular uptake of lysozyme-stabilized gold nanoparticles]
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* Stock solutions of 5 mL 4.36 mM HAuCl<sub>4</sub> and 5 mL 5 mM lysozyme both in deionized water were made.
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** Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/10/23|Dhea Patel's entry]] for theoretical and actual concentration calculations of the lysozyme stock solution, as well as the protocol for preparing Au-lysozyme solutions.
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  '''MW HAuCl<sub>4</sub>: 339.785 g/mol'''
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  '''Actual mass HAuCl<sub>4</sub>: 7.4 mg'''
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  '''Volume H<sub>2</sub>O added: 5 mL'''
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  '''[(0.0074 g HAuCl<sub>4</sub>)/(339.785 g/mol)]/0.005 L = 0.00436 M HAuCl<sub>4</sub>'''
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Objectives

  • Make stock solutions of luminol, H2O2, carbonate buffer, and HRP for optimizing the HRP-luminol assay.
  • Make 7 8 mL solutions of Au-lysozyme at mole ratios of 10, 15, 20, 25, 30, 35, and 40.

HRP-Luminol Assay

10 mL 0.5 mM luminol stock solution in carbonate buffer:

  • Theoretical: 0.0008858 g luminol in 10 mL carbonate buffer gives a 0.5 mM luminol solution.
  • Actual: 0.0010 g luminol in 10 mL carbonate buffer gives a 0.564 mM luminol solution.


10 mL 5 mM H2O2 stock solution in carbonate buffer:

  • Theoretical: 3.9 μL of 30 wt% H2O2 in 10 mL of carbonate buffer gives a 5 mM H2O2 solution.
  • Actual: As automated pipetters were used to create the solution, there was no deviation between theoretical and actual results.


1 mL 9.2 μM HRP in autoclaved water:

  • Theoretical: 4.0 mg HRP in 1 mL autoclaved water gives a 9.2 μL HRP solution.
  • Actual: There were no deviations from the theoretical results.


100 mL 1 mM sodium carbonate in water:

  100 mL 1 mM sodium carbonate solution = 1 × 104 mol
  MW sodium carbonate: 105.9784 g/mol
  (1 × 104 mol)×(105.9784 g/mol) = 0.01060 g sodium carbonate
  • Theoretical: Add 0.01060 g sodium carbonate to 100 mL of water to give a 1 mM sodium carbonate solution.
  • Actual: 0.01060 g of sodium carbonate was added to 100 mL of water.


100 mL 1 mM sodium bicarbonate in water:

  100 mL 1 mM sodium bicarbonate solution = 1 × 104 mol
  MW sodium carbonate: 84.007 g/mol
  (1 × 104 mol)×(84.007 g/mol) = 0.00840 g sodium bicarbonate
  • Theoretical: Add 0.00840 g of sodium bicarbonate to 100 mL of water to give a 1 mM sodium bicarbonate solution.
  • Actual: 0.00840 g of sodium bicarbonate was added to 100 mL of water.


Adjusting the pH of Carbonate Buffer to 8.5

  • The initial pH of 100 mL of 1 mM sodium carbonate solution was measured to be 10.05.
  • Drops of 1 mM sodium bicarbonate solution were added to the sodium carbonate solution until the pH reached 8.5. A total volume of 20 mL of 1 mM sodium bicarbonate solution was used, such that the total volume of the solution was 120 mL.
  • The final concentration of sodium carbonate in the pH-adjusted buffer was 0.833 mM.
  Total volume of solution: 120 mL
  Number of moles of sodium carbonate in the original solution: 1 × 104 mol
  New concentration of sodium carbonate: (1 × 104 mol)×(0.120 L) = 0.833 mM

Au-Lysozyme Solutions

  MW HAuCl4: 339.785 g/mol
  Actual mass HAuCl4: 7.4 mg
  Volume H2O added: 5 mL
  [(0.0074 g HAuCl4)/(339.785 g/mol)]/0.005 L = 0.00436 M HAuCl4



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