User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/11/14: Difference between revisions
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==Goals== | ==Goals== | ||
* We picked up from yesterday and continued with the [[AU Biomaterials Design Lab:Protocols/ADA Activity Assay| Kinetic Assay protocol]] | * We picked up from yesterday and continued with the [[AU Biomaterials Design Lab:Protocols/ADA Activity Assay| Kinetic Assay protocol]] | ||
* We made solutions of gold in our sample of adenosine deaminase. | * We made solutions of gold in our sample of adenosine deaminase. This procedure and concentrations can be found in [[User:Michael F. Nagle/Notebook/Chem 571/2012/11/14| Michael's notebook]] | ||
* We also began the process of conducting dialysis on our samples of ADA in order to remove the salts from the samples | * We also began the process of conducting dialysis on our samples of ADA in order to remove the salts from the samples | ||
==Procedure== | ==Procedure== | ||
* For all information regarding the | * For all information regarding the procedure followed for the dialysis of the ADA, please see [[User:Michael F. Nagle/Notebook/Chem 571/2012/11/14| Michael's notebook.]] | ||
* From the spectras collected yesterday, we looked at the absorbances of inosine and adenosine at two specific wavelengths, 235nm and 265 nm. These absorbances were used to calculate the molar absorptivity using Beers Law A=εbc | * From the spectras collected yesterday, we looked at the absorbances of inosine and adenosine at two specific wavelengths, 235nm and 265 nm. These absorbances were used to calculate the molar absorptivity using Beers Law A=εbc | ||
** For inosine, at λ235, ε= 1400; λ265 ε= 6000 | ** For inosine, at λ235, ε= 1400; λ265 ε= 6000 | ||
** and for Adenosine at λ235, ε= 18566; λ265 ε= 13029 | ** and for Adenosine at λ235, ε= 18566; λ265 ε= 13029 | ||
(For full calculations, please see Mary's notebook) | (For full calculations, please see [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/11/14| Mary's notebook]]) | ||
** based on these calculations, which were checked multiple times, the greatest differential was seen to be at λ235, where adenosine has an extremely high molar absorptivity and comparatively, the absorptivity for inosine was relatively low. Based on this fact, in the following samples, we monitored the kinetics of the reaction at a wavelength of 235. in these samples, there was adenosine, ADA and phosphate buffer present. in the catalysis of this reaction, ADA being an enzyme would eat up the adenosine and convert it into inosine. as a result, on our spectra, we expected to see the absorbance of the sample to decrease as time went by. | ** based on these calculations, which were checked multiple times, the greatest differential was seen to be at λ235, where adenosine has an extremely high molar absorptivity and comparatively, the absorptivity for inosine was relatively low. Based on this fact, in the following samples, we monitored the kinetics of the reaction at a wavelength of 235. in these samples, there was adenosine, ADA and phosphate buffer present. in the catalysis of this reaction, ADA being an enzyme would eat up the adenosine and convert it into inosine. as a result, on our spectra, we expected to see the absorbance of the sample to decrease as time went by. | ||
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**Inosine: λ235, ε= 7540; λ265 ε= 5530 | **Inosine: λ235, ε= 7540; λ265 ε= 5530 | ||
** This new data shows that what we had been measuring the entire time at the 235nm wavelength was the absorbance of the inosine which was supposed to increase with the addition of the ADA. As a result, we will be continuing with this portion of the experiment in the future. | |||
Revision as of 11:00, 7 December 2012
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Continue ADA kinetics assayGoals
Procedure
(For full calculations, please see Mary's notebook)
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