User:Melissa Novy/Notebook/CHEM-571/2012/09/26

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Revision as of 15:09, 7 October 2012 by Abigail E. Miller (talk | contribs) (Objectives)
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Objectives

  • Prepare cells for protein expression.
    • Make binding buffer and elution buffer.
    • Please refer to Dhea Patel's entry for the protocols and calculations of the above solutions.
  • Abigail E. Miller 18:09, 7 October 2012 (EDT):you need ot include the specifics of the bidnign nad elution bufers, pH final volume and concnetratiosn of each component.
  • Continue optimizing the HRP chemiluminescence assay by adjusting the concentrations of the reactants.
  • Analyze Tris buffers at pH 8 and 10 and at various concentrations with UV-vis spectroscopy.

Spectroscopy Data of Tris Buffers

Tris Buffer Absorbance.JPG

  • Graph of absorbance versus wavelength of Tris buffer in H2O at pH 8 and 10 and at concentrations ranging from 10 μM to 10 mM.
  • Note that very small peaks were observed around 730-740 nm for all solutions and another small, broad peak was observed around 600 nm for the 10 μM Tris buffer solution at pH 8. The data indicate that absorbance of Tris buffer does not affect the peak observed for AuNPs at around 540 nm and the concentration and pH of Tris does not significantly affect the peaks for Tris observed.

HRP Chemiluminescence Assay

  • Concentrations of solutions tested:
Trial Phenol Luminol H2O2 HRP
1 18 mM 1.25 mM 1.7 mM 0.23 µM
2 18 mM 0.625 mM 1.7 mM 0.23 µM
3 18 mM 1.25 mM 1.7 mM 9.2 µM
4 18 mM 1.25 mM 1.7 mM 4.6 µM


HRP Chemiluminescence Assay.JPG

  • Graph of intensity versus time of 4-iodophenol-enhanced solutions of hydrogen peroxide, luminol, and HRP.
  • Refer to the report for analysis of HRP assays with AAP and luminol.