User:Dhea Patel/Notebook/Experimental Biological Chemistry Notebook/2012/11/27

Objective

• to analyze the AuHRP and AuADA solutions made on November 14th using both UV-vis and AAS.
• to prepare AuADA solutions using dialyzed ADA (the ADA was dialyzed from November 14th until November 27th).
• to resuspend AuHRP fibers in Tris Buffer

Description

Analyzing AuHRP and AuADA Mixtures

• UV-vis
  • 3mL of each sample were pipetted using a Pipetman into a quartz cuvette and placed in the UV-vis.
  • The data was collected, corrected for water, and plotted. Please see below under "Data" for the graph.
• AAS
  • HCl was used as a blank and the AAS was auto-corrected for water.
  • 8 standards were used: 5ppm Au, 10ppm Au, 15pp Au, 20ppm Au, 25ppm Au, 30ppm Au, 35ppm Au, and 40ppm Au.
  • Each sample of AuHRP and AuADA was run and the measured concentration was collected and plotted.

"Preparing AuADA solutions"

• Please refer to Kay's entry for details on removing the ADA from the snake-skin dialysis tubing and preparing the AuADA solutions.
  • Note that in preparing the AuADA solutions, the only difference in preparation from that of November 14th was the use of dialyzed ADA, rather than the purified ADA solution.

"Resuspension of Protein Fibers in Tris Buffer"

• Please refer to Kay's entry for details on fiber resuspension, including Tris buffer volumes and concentrations.
  • Tris buffer was added to 130, 140, and 150 molar ratio Au:HRP fibers and allowed to sit over night.

Data/Observations

• The spectra show a consistent peak at 520 nm, indicating the presence of AuNPs.
• There was a direct relationship between the absorbance and the mole ratio of Au:HRP. As the mole ratio increased, the absorbance increased.
• The 130 AuHRP solution contained 1 mL less of H2O due to an error during preparation of the solution, which may account for the similarity in absorbance peake for 130 and 140 mole ratio Au:HRP solutions.

• No peaks were formed in this spectra, indicating the absence of AuNPs.
• The solutions themselves were colorless, so these results were expected.
• The ADA used in these solutions was pre-dialysis, so the ADA was in elution buffer containing high levels of imidazole, which may have interfered with the AuNP formation.

• This table shows the absorbance values of each of the standard used for AAS.

• This table lists the absorbance of each sample and its concentration in ppm.