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

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  • 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]
  • 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 both 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.
  • After measurement, each 3mL samples of Au/ADA and Au/HRP of different mole ratios were placed back into original sample test tubes to conserve products that would later be used for Atomic Absorption spectra analysis.

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

  • Au/ADA samples appeared clear, and no purple fibers were formed. This also indicates that no gold nanoparticles were present in solution. However, there were white precipitants observed at the bottom of the samples in samples with molar ratio 130 to 150. The white precipitants appear in small circular quantities and seemed to stick to the side of the glass test tube. A picture of white precipitant in Au/ADA with 150 mole ratio is shown below:

  • It was suspected that the white precipitant could be precipitated ADA proteins when gold interacts with the salt in elution buffer. The elution buffer contained the following contents: 20mM Tris, 0.5M NaCl, and 500mM Imidazole at pH 7.5. Once salt is attracted to interact with HAuCl4 in solution, ADA protein then precipitates due to lack of buffering for stabilization.
  • Au/HRP samples appeared purple in color. All samples had purple supernatant. Small fibers were observed for sample with mole ratio 130 to 150. For samples with 100 to 150 molar ratio, supernatant displayed a darker purple color compare to samples with 60 to 90 molar ratio.
  • UV-vis spectrometer were run for Au/ADA samples. Absorbance versus wavelength ranging from 200nm to 800nm were plotted on the graph titled "Absorbance of AuADA Solutions at Mole Ratios from 60 to 140 Au:ADA", shown below:

  • From graph above, it can be shown that Au/ADA samples have very low absorbance. This indicates that litle to none gold nanoparticles were formed within solution. It was also suspected a potential peak around 700nm from seeing the overall trend for all samples. However, that peak would most likely be due to the salt present in elution buffer.
  • UV-vis spectrometer were run for Au/HRP samples. Absorbance were graphed versus wavelength for samples with mole ratio ranging from 60 to 150. The graph titled "Absorbance of Solutions of AuHRP at Mole Ratios from 60 to 150 Au:HRP" is shown below:

  • From the graph, it can be suggested that 150 Au/HRP solution yielded the most amount of absorbance. This result seemed consistent to the procedure for making Au/HRP solutions since most amount of HAuCl4 was placed into the 150 Au/HRP sample.
  • The results from UV-vis spectra also suggested gold nanoparticles were present in all solutions. Maximum concentrations of gold nanoparticles were present in 150 Au/HRP samples while 60 Au/HRP sample contained the least concentrations of gold nanoparticles.
  • Wavelength at 525nm was looked specifically because it was the wavelength that yielded the maximum amount of absorbance. Table listed below shows the absorbance at 525nm for all samples. The column on the right showed corrected absorbance with absorbance of water subtracted.
Au/HRP mole ratio Absorbance at 525nm Absorbance without Water
  • A graph with absorbance plotted against the increasing mole ratio of Au/HRP is shown below. Absorbance used in graph was the corrected absorbance.

  • From the graph above, it could be suspected that there is a linear relationship between the mole ratio of Au to HRP in solution and the concentrations of gold nanoparticles in solution before protein aggregation takes over.

Procedure for Running AA Spectrometer on Au/ADA and Au/HRP samples

  • Atomic Absorption spectra were first calibrated with HCl and gold solutions with the following range of concentrations:
  5ppm - 8ppm - 10ppm - 15ppm - 20ppm - 25ppm - 30ppm - 40ppm
  • Once the AA spectrometer was warmed up, the above HCl and gold solutions were run one by one for calibration. After the above eight solutions have ran, instrument was successfully calibrated.
  • Each Au/ADA and Au/HRP samples were injected into the AA spectrometer and ran individually. In between each run, the AA spectrometer inject tube was rinsed with distilled water.
  • The absorbance for Au/ADA and Au/HRP samples were recorded and converted to ppm.

Results for Running AA Spectrometer on Au/ADA and Au/HRP samples

  • Standard HCl samples contain different concentrations of gold were tested and were used to calibrate the Atomic Absorption Spectrometer. The absorbance for each HCl standards are shown below:
Standard HCl sample Concentration of HCl [ppm] Absorbance(Abs.)
  • Au/HRP were run with the above calibration, and a table listing the absorbance and calculated concentration in units of ppm is shown below:
Au/HRP samples Absorbance(Abs.) Concentration[ppm]
  • A graph was plotted with concentration of gold in Au/HRP versus Au/HRP mole ratios shown below:

  • From the graph, it can be seen that as the mole ratio of Au to HRP in solution, the concentration of gold in solution also increases. If the data point at 130 Au/HRP with concentration 16.5305ppm is eliminated, then one can conclude that there is a positive linear relationship between the mole ratio of Au/HRP and concentration of gold in ppm in solution before nanoparticles in solutions starts to form aggregations.
  • Au/ADA samples ranging from mole ratio of 60 to 150 were also run on Atomic Absorption spectrometer. Each samples with the corresponding absorbance and calculated concentration in ppm are shown in table below:
Au/ADA samples Absorbance(Abs.) Concentration[ppm]
  • Concentration of gold in ppm was plotted against the mole ratio of Au to ADA ranging from 60 to 150. The graph titled "Concentration of Gold in Au/ADA [ppm] versus Au/ADA Mole Ratios" is shown below:

  • From the graph above, it can be concluded that as the mole ratio of Au to ADA increases, the concentration of gold in solution also increases. However, in the above data, there was an outlier at 100 Au/ADA with the calculated concentration 5.2261ppm. This outlier might be due to a lack of raising before sample 100 Au/ADA was injected, leaving more gold residue to be analyzed compare to before.
  • It can be seen that the concentration of gold in solution in Au/HRP appeared almost double as the concentration of gold in Au/ADA solutions. This result could be explained with following reasons:
    • The HRP is more flexible in structure and thus can lead to more HRP folding within 85°C and 4 hours of heating. The ADA might have a less flexible structure, and might be limited with the presence of more beta sheets within the protein structure.
    • The HRP molecules has a larger molecular weight (44kDa) compare to the molecular weight of ADA (41kDa). The larger molecular weight might have a larger surface area to interact with gold in solution when heating. More gold nanoparticles might be formed due to the exposure between protein surface area and gold in solution in Au/HRP solutions.
  • Both Au/HRP and Au/ADA solution exhibit positive linear relationship between increased mole ratio and concentration of gold in solution. This suggested that both enzyme undergoes similar mechanism when interacting with HAuCl4 under heat.

Procedure for Fiber resuspension in Tris buffer

  • Au/HRP samples with mole ratio 130 to 150 were used to test fiber resuspension with Tris buffer.
  • Tris buffer with the following concentrations were used for test resuspension:
  • A table was made to list the Tris buffer used for resuspension in Au/HRP samples:
Tris Buffer used Au/HRP samples used Amount of Buffer added
10mM at pH 10130 Au/HRP1mL
50mM at pH 10140 Au/HRP1mL
500mM at pH 10150 Au/HRP1mL
  • The 1mL tris buffer were inserted into samples and pipetted up and down to mix until no fibers were observed.
  • After resuspension, the solution with added buffer was left in room temperature. The resuspension was checked after 24 hours for possibilities of fiber precipitation.

Results for Fiber resuspension in Tris buffer

  • The fibers were resuspended fully after pipetting solution up and down to mix. A picture of resuspended solution is shown below:

  • After 24 hours, no purple fibers were formed, thus it can be concluded that the addition of salt into solution interacts with gold nanoparticle aggregation in a way that disrupts the aggregation. The lack of precipitation after 24 hours can be due to the gold nanoparticles being more stable when interacting with Tris buffer.
  • A purple homogenous solution was yielded after the addition of Tris buffers at all three concentrations used. Thus, it can be safe to conclude that Tris buffer at pH 10.0 can successfully resuspend fibers at a 6:1 ratio of solution to tris buffer.
  • Although the exact mechanism for resuspension is unknown, successful resuspension might be explained with following reasoning:
    • The presence of salt into the solution disrupts the protein aggregation and stabilizes the disrupted gold nanoparticles with surrounding salts in solution.
    • The isoelectric point for the protein aggregation might cause the aggregation to interact with the positive and negative charge of the salt in solution. After aggregation is disrupted, pH 10 of the protein allowed gold nanoparticles to be stable in solution.
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