User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/27

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Purpose

  • ADA protein were transferred from dialysis tubing into 15mL falcon tubes
  • Make Au/ADA samples with the following mole ratios: 60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150, with ADA fraction 2 after dialysis.
  • Un-dialyzed Au/ADA samples and Au/HRP made on 2012/11/14 were run on UV-vis spectrometer and Atomic Absorption Spectrometer in order to compare spectra results for Au/ADA, Au/HRP, Au/Lysozyme, and Au/BSA.
  • Resuspend Au/HRP samples in different concentration and pH of Tris buffer to test ionic strength.

Procedure for Dialysis

  • Dialysis beaker containing dialysis tubing enclosed with ADA protein fractions were taken from the 4°C cold room into room temperature lab room.
  • The dialysis clips were taken off from dialysis tubing. ADA protein fractions were poured into a sterile 15mL falcon tubes.
  • This process was repeated for all three protein fractions: ADA fraction 1&3 made on 2012/11/06, ADA fraction 2 made on 2012/11/06, and ADA fraction purified on 2012/10/03.
  • ADA fractions in 15mL falcon tubes were stored in 4°C refrigerator.

Notes for Dialysis

  • It was observed that white precipitants were found at the bottom of dialysis tubing, and suspected that due to a lack of salt in solution with ADA proteins, the ADA proteins falls out of solution. This could predict a potential difference in UV-vis and Atomic Absorption spectrometer results in finalized product of Au/ADA samples made with dialyzed ADA compare to Au/ADA samples made on 2012/11/14.

Procedure for making dialyzed Au/ADA samples

  • Au/ADA samples were made with the following mole ratios:
  60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150
  • Stock solution of HAuCl4 was made on 2012/09/05 with a concentration of 10.5uM.
  • Stock solution for ADA was the ADA protein fraction made in 2012/10/03 after dialysis. The ADA stock solution has a concentration of 58.36μM.
  • Volume of ADA protein was set at 137.1uL and a range of HAuCl4 was used from 45.71μL to 114.3μL. Water was added to the sample to increase the volume of sample to 8mL. Volumes of each reactants are shown in table below:
Au/ADA ratio ADA added[uL] HAuCl4 Added [uL] Water Added[uL] [ADA]final[uM] [HAuCl4]final[uM]
60137.145.717817.2160
70137.153.37809.6170
80137.160.97802180
90137.168.67794.4190
100137.176.27786.81100
110137.183.87779.11110
120137.191.47771.51120
130137.1997763.91130
140137.1106.77756.31140
150137.1114.37748.71150
  • The samples were made in 15mL non-sterile falcon tubes. After all reactants were added, samples are capped and wrapped around with aluminum foil.
  • Samples were placed in incubator at 85°C for 4 hours then cooled down to room temperature.

Procedure for Running UV-vis Spectrometer on Au/ADA and Au/HRP samples

  • UV-vis spectrometer were run in spectrum method. A range of 200nm to 800nm were ran on all Au/ADA and Au/HRP samples made on 2012/11/14.
  • 3mL of distilled water was placed in quartz cuvette with 1cm pathlength and set as base line solution. 3mL taken from supernatant of Au/ADA and Au/HRP samples were placed in an identical quartz cuvette with 1cm pathlength for spectra measurement.

Results for Running UV-vis Spectrometer on Au/ADA and Au/HRP samples


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