User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/27: Difference between revisions
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* 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 were run with the above calibration, and a table listing the absorbance and calculated concentration in units of ppm is shown below: | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Au/HRP samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Absorbance(Abs.)''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration[ppm]''' | |||
|- | |||
| 60||0.0827||3.8088 | |||
|- | |||
| 70||0.1042||5.2282 | |||
|- | |||
| 80||0.1102||5.6243 | |||
|- | |||
| 90||0.1428||7.7765 | |||
|- | |||
| 100||0.1584||8.8064 | |||
|- | |||
| 110||0.166||9.3081 | |||
|- | |||
| 120||0.1901||10.8992 | |||
|- | |||
| 130||0.2754||16.5305 | |||
|- | |||
| 140||0.2144||12.5034 | |||
|- | |||
| 150||0.2473||14.6754 | |||
|} | |||
* A graph was plotted with concentration of gold in Au/HRP versus Au/HRP mole ratios shown below: | |||
[[Image:Au HRP gold.png|350px]] | |||
* 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: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Au/ADA samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Absorbance(Abs.)''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration[ppm]''' | |||
|- | |||
| 60||0.0417||1.1021 | |||
|- | |||
| 70||0.0571||2.1188 | |||
|- | |||
| 80||0.0605||2.3432 | |||
|- | |||
| 90||0.0761||3.3731 | |||
|- | |||
| 100||0.1041||5.2261 | |||
|- | |||
| 110||0.0828||3.8154 | |||
|- | |||
| 120||0.0854||3.9871 | |||
|- | |||
| 130||0.102||5.083 | |||
|- | |||
| 140||0.112||5.7431 | |||
|- | |||
| 150||0.116||6.0072 | |||
|} | |||
* 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: | |||
[[Image:Au ADA gold.png|350px]] | |||
* 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 HAuCl<sub>4</sub> 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: | |||
** 10mM Tris buffer at pH 10.0 made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/05|2012/09/05]] | |||
** 50mM Tris buffer at pH 10.0 made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/12|2012/09/12]] | |||
** 500mM Tris buffer at pH 10.0 made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/09/12|2012/09/12]] | |||
* A table was made to list the Tris buffer used for resuspension in Au/HRP samples: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Tris Buffer used''' | |||
| align="center" style="background:#f0f0f0;"|'''Au/HRP samples used''' | |||
| align="center" style="background:#f0f0f0;"|'''Amount of Buffer added''' | |||
|- | |||
| 10mM at pH 10||130 Au/HRP||1mL | |||
|- | |||
| 50mM at pH 10||140 Au/HRP||1mL | |||
|- | |||
| 500mM at pH 10||150 Au/HRP||1mL | |||
|} | |} | ||
* 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: | |||
[[Image:Photo13.jpg|400px]] | |||
* 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. |
Revision as of 13:29, 28 November 2012
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Purpose
Procedure for Dialysis
Notes for Dialysis
Procedure for making dialyzed Au/ADA samples
60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150
Procedure for Running UV-vis Spectrometer on Au/ADA and Au/HRP samples
Results for Running UV-vis Spectrometer on Au/ADA and Au/HRP samples
Procedure for Running AA Spectrometer on Au/ADA and Au/HRP samples
5ppm - 8ppm - 10ppm - 15ppm - 20ppm - 25ppm - 30ppm - 40ppm
Results for Running AA Spectrometer on Au/ADA and Au/HRP samples
Procedure for Fiber resuspension in Tris buffer
Results for Fiber resuspension in Tris buffer
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