User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/13: Difference between revisions
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* It was concluded to test ratios between 50 to 150 in smaller increments for more accurate conclusion on when protein aggregates form. | * It was concluded to test ratios between 50 to 150 in smaller increments for more accurate conclusion on when protein aggregates form. | ||
==Procedure on Atomic Absorption Spectrometry of Au/ | ==Procedure on Atomic Absorption Spectrometry of Au/lysozyme and Au/HRP solutions== | ||
* 8 of HCl samples with the concentrations below were run to set up calibration curve: | |||
5ppm - 8ppm - 10ppm - 15ppm - 20ppm - 25ppm - 30ppm - 40ppm | |||
* The absorbance obtained were graphed with absorption versus concentration of HCl. The slope of the plot was used to calculate the concentration of gold in Au/HRP and Au/lysozyme solutions. Because the absorption data and graph were not saved during experimental process, the results cannot be displayed in Data section. | |||
* Supernatant of Au/HRP samples made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/07|2012/11/07]] with a ratio ranging from 10 to 450 were run using Atomic Absorption Spectrometry. | |||
* Supernatant of Au/lysozyme samples made on [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/31|2012/10/31]] with a ratio ranging from 20 to 130 were also run using Atomic Absorption Spectrometry. | |||
* Distilled water was prepared in a 280mL flask to rinse off samples after Au/HRP and Au/lysozyme samples were run. | |||
* Samples with different ratios of gold to either HRP or lysozyme was run by inserting sample tube into the supernatant of samples. The purple fibers in the samples were avoided to prevent clogging. After the absorbance of samples were taken, the sample tubes were inserted into distilled water for rinsing. This process was repeated until all samples were run. | |||
* The absorbance for each Au/HRP and Au/lysozyme samples were collected by the atomic absorbance spectrometer. The absorbance for each sample were fitted into the slope of calibration curve to obtain the concentration of gold in unit of ppm. Because the raw data were lost, the numbers cannot be shown here. | |||
==Data on Atomic Absorption Spectrometry of Au/ADA and Au/HRP solutions== | |||
* The concentration of gold in unit of ppm was calculated by converting absorbance into concentration using the HCl calibration curve. | |||
* The concentration of gold for corresponding ratios of Au/HRP and Au/Lysozyme were displayed in table below: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Au/HRP ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration of Au[ppm]''' | |||
| align="center" style="background:#f0f0f0;"|'''Au/Lysozyme Ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration of Au[ppm]''' | |||
|- | |||
| 10||-1.5443||20||0.0057 | |||
|- | |||
| 50||1.8191||30||1.9388 | |||
|- | |||
| 100||11.7714||40||2.7586 | |||
|- | |||
| 150||13.1419||50||3.806 | |||
|- | |||
| 200||23.6748||60||4.5002 | |||
|- | |||
| 250||24.4588||70||-2.4599 | |||
|- | |||
| 300||27.876||80||-2.4839 | |||
|- | |||
| 350||25.4761||100||-2.5856 | |||
|- | |||
| 400||35.1173||120||-2.6394 | |||
|- | |||
| 450||25.6198||130||-2.6454 | |||
|} | |||
* A graph was made with concentration of gold in ppm in supernatant versus mole ratio of Au to HRP and displayed below: | |||
[[Image:AAS_AuHRP.JPG|600px]] | |||
* From the graph above, increase in mole ratio of Au/HRP leads to an linear trend of increase in gold concentration in supernatant. This indicates no protein aggregation would form as mole ratio of Au/HRP increases. The result provide a contrast with results from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/17|2012/10/17]] with the maximum concentration of gold in supernatant at low mole ratios of Au/BSA and minimum concentration of gold in supernatant once the mole ratios of Au/BSA reaches over 134. | |||
* The result indicate that protein aggregation is less likely to form when HRP is used to replace BSA. This might be caused by the nature of HRP compare to BSA and different behaviors when proteins unfold and refold. | |||
* Gold concentrations in Au/Lysozyme samples were also determined and plotted onto the graph "Concentration of Au in Solution of Au-Lysozyme" below: | |||
[[Image:AAS_AuLys.JPG|600px]] | |||
* In the graph above, the concentration of gold in Au/Lysozyme supernatant increased from mole ratio of 20 to 60, and dropped immediately from mole ratio of 60 and on. From mole ratio of 60 and on, there are no gold present in supernatant. The samples with mole ratio from 60 and on appear transparent as oppose to purple. | |||
* This result indicates that protein started to form aggregation from mole ratio of 60 and on. Compare to the result with Au/HRP from above and Au/BSA from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/17|2012/10/17]], the behavior of Au/Lysozyme resemble more like Au/BSA samples in terms of protein aggregation formation. This result also indicates that BSA and lysozyme behavior similarly when forming gold nano particles. It can also indicate that they have similar protein folding and unfolding kinetic mechanism. | |||
==Procedure on making adenosine== | |||
* Adenosine free base was obtained in powder form from aMR-ESCO®. | |||
* Sodium phosphate dibasic Heptahydrate was obtained from Fisher Scientific®. | |||
* Diluted adenosine in liquid form was made to prepare for ADA activity assay. | |||
* 0.05M sodium phosphate buffer was made in 50mL of distilled water. | |||
* The pH of sodium phosphate buffer was adjusted to 7.4 using HCl. | |||
* 0.1mM of adenosine was made in 5mL of sodium phosphate buffer. | |||
* This procedure was not done by writer, thus was not described in details. For details of the calculations, see [[User:Melissa Novy/Notebook/CHEM-571/2012/11/13|Melissa's Notebook]], which includes calculations for making sodium phosphate buffer, pH adjustment of sodium phosphate buffer, calculations to weight out the adenosine, and calculations on the amount of sodium phosphate buffer to add to adenosine in order to reach 0.1mM adenosine. | |||
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Purpose
Procedure on UV-vis spectrometry of Au/HRP
10 - 50 - 100 - 150 - 200 - 250 - 300 - 350 - 400 - 450
Data on UV-vis spectrometry of Au/HRP
Procedure on Atomic Absorption Spectrometry of Au/lysozyme and Au/HRP solutions
5ppm - 8ppm - 10ppm - 15ppm - 20ppm - 25ppm - 30ppm - 40ppm
Data on Atomic Absorption Spectrometry of Au/ADA and Au/HRP solutions
Procedure on making adenosine
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