User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/02

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(Preparation of the Reactants of HRP Oxidase Assay)
(Preparation of the Reactants of HRP Oxidase Assay)
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* To obtain 0.0017 M of hydrogen peroxide, added 1 mL 30% of the clear and colorless liquid into 100 mL of water. Then, collected 1 mL from the solution to dilute it into 50 mL of the 0.2 M phosphate buffer.   
* To obtain 0.0017 M of hydrogen peroxide, added 1 mL 30% of the clear and colorless liquid into 100 mL of water. Then, collected 1 mL from the solution to dilute it into 50 mL of the 0.2 M phosphate buffer.   
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* In reference to the document of [http://openwetware.org/wiki/Image:HRP_Assays.docx Novy, Patel, and Wang]

Revision as of 00:13, 8 October 2012

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Preparation of the Reactants of HRP Oxidase Assay

  • The reactants listed in HRP Oxidase Assay were weighed out.
  • The phosphate buffer was made with white granules of sodium phosphate dibasic (FW 268.07, heptahydrate) instead of potassium phosphate. The substitution is not detrimental to the procedure since the ion of interest is the phosphate group. The total volume chosen for the buffer is 60 mL.
  • Calculation for obtaining Na2HPO4:

0.020 mol of Na2HPO4 × \frac{268.07g}{1mol} of Na2HPO4 = 53.61 g of Na2HPO4


x60 mL = \frac{.060 L * 53. 61 g}{1L} = 3.22 g of Na2HPO4


  • The required weight for 4-iodophenol is 0.810 g to make 0.0025 M. The actual weighed amount was 0.8109 g. This amount is dissolved in 700 μM dimethyl sulfoxide (DMSO, polar aprotic solvent) due to its immiscibility in water.
  • Weighed out 1 mg of the brown granular solid of horseradish peroxidase (HRP) and dissolved it in 1 mL water.
  • To obtain 0.0017 M of hydrogen peroxide, added 1 mL 30% of the clear and colorless liquid into 100 mL of water. Then, collected 1 mL from the solution to dilute it into 50 mL of the 0.2 M phosphate buffer.



  • Abigail E. Miller 10:40, 7 October 2012 (EDT):you are missing a bunch or reactants and your data from this date. show what happened and what problem you had to assess for the next day.



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