User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/11/18: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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### 90min | ### 90min | ||
### 120 min | ### 120 min | ||
### NOTE: We made two of each of the samples so that we could average the absorbance values of both samples for each time interval | |||
## Next, we determined how much of our protease stock we should add to each of the sample tubes in order to make the final concentration of alpha-chymotrypsin 1µM and the final volume of the sample 1mL: | ## Next, we determined how much of our protease stock we should add to each of the sample tubes in order to make the final concentration of alpha-chymotrypsin 1µM and the final volume of the sample 1mL: | ||
### We did the following calculation to determine how much of the alpha-chymotrypsin stock we would need to add to each sample and blank to bring the final concentration of alpha-chymotrypsin to 1nM in each:<br>Let<br>M<sub>1</sub>=concentration of alpha-chymotrypsin stock = 74.21875µM<br>V<sub>1</sub>=volume of protease stock needed<br>M<sub>2</sub>=concentration of alpha-chymotrypsin needed in the sample tube = 1µM<br>V<sub>2</sub>=volume of the solution in the sample tube = 1mL<br>M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub><br>V<sub>1</sub>=(M<sub>2</sub>V<sub>2</sub>)/(M<sub>1</sub>)<br>V<sub>1</sub>=((1µM)(1mL))/(74.21875µM)<br><b>V<sub>1</sub>=0.01347mL=13.47µL</b> | ### We did the following calculation to determine how much of the alpha-chymotrypsin stock we would need to add to each sample and blank to bring the final concentration of alpha-chymotrypsin to 1nM in each:<br>Let<br>M<sub>1</sub>=concentration of alpha-chymotrypsin stock = 74.21875µM<br>V<sub>1</sub>=volume of protease stock needed<br>M<sub>2</sub>=concentration of alpha-chymotrypsin needed in the sample tube = 1µM<br>V<sub>2</sub>=volume of the solution in the sample tube = 1mL<br>M<sub>1</sub>V<sub>1</sub>=M<sub>2</sub>V<sub>2</sub><br>V<sub>1</sub>=(M<sub>2</sub>V<sub>2</sub>)/(M<sub>1</sub>)<br>V<sub>1</sub>=((1µM)(1mL))/(74.21875µM)<br><b>V<sub>1</sub>=0.01347mL=13.47µL</b> | ||
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<b><center>Figure 1: Absorbance of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)</center></b> | <b><center>Figure 1: Absorbance of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)</center></b> | ||
[[ | [[Image:20151118 bonan bradford blanks.png|thumb|center|700px]] | ||
The above figure shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The absorbance was corrected by first subtracting the absorbance of a Bradford blank<sup>*</sup> from the absorbance each data point at their respective wavelengths. The absorbance was then corrected by subtracting the absorbance at the isosbestic point of each sample and blank from all of the absorbance values for the respective sample and blank. | The above figure shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The absorbance was corrected by first averaging the raw absorbance data for each duplicate sample and then subtracting the absorbance of a Bradford blank<sup>*</sup> from the absorbance of each averaged data point at their respective wavelengths. The absorbance was then corrected by subtracting the absorbance at the isosbestic point of each sample and blank from all of the absorbance values for the respective sample and blank. | ||
*NOTE: The blank was just Bradford reagent and Tris buffer. | *NOTE: The blank was just Bradford reagent and Tris buffer. | ||
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<b><center>Figure 2: Absorbance of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)</center></b> | <b><center>Figure 2: Absorbance of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)</center></b> | ||
[[ | [[Image:20151118 bonan bradford samples.png|thumb|center|700px]] | ||
The above figure shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance was corrected in the same way that the absorbance for the alpha-chymotrypsin blanks was corrected. | The above figure shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance was corrected in the same way that the absorbance for the alpha-chymotrypsin blanks was corrected. | ||
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<b><center>Figure 3: Absorbance of alpha-Chymotrypsin Blanks and AuNP Fiber Samples at 600nm as a Function of Incubation Time (min)</center></b> | <b><center>Figure 3: Absorbance of alpha-Chymotrypsin Blanks and AuNP Fiber Samples at 600nm as a Function of Incubation Time (min)</center></b> | ||
[[ | [[Image:20151118 bonan bradford blanksnsamples.png|thumb|center|700px]] | ||
The above figure shows the absorbance of the alpha-chymotrypsin blanks and the AuNP fiber samples at 600nm as a function of the amount of time that they were incubated. The absorbance values are taken directly from the absorbances in Figures 1 and 2. | The above figure shows the absorbance of the alpha-chymotrypsin blanks and the AuNP fiber samples at 600nm as a function of the amount of time that they were incubated. The absorbance values are taken directly from the absorbances in Figures 1 and 2. | ||
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<b><center>Figure 4: Absorbance of AuNP Fiber Samples-alpha-Chymotrypsin Blanks at 600nm as a Function of Incubation Time (min)</center></b> | <b><center>Figure 4: Absorbance of AuNP Fiber Samples-alpha-Chymotrypsin Blanks at 600nm as a Function of Incubation Time (min)</center></b> | ||
[[ | [[Image:20151118 bonan bradford peptides.png|thumb|center|700px]] | ||
The above figure shows the absorbance of the AuNP fiber samples (after subtracting out the absorbance of the alpha-chymotrypsin blanks) at 600nm as a function of incubation time. It effectively shows the absorbance of the peptides and AuNP that had gone into solution as a result of degradation by alpha-chymotrypsin. | The above figure shows the absorbance of the AuNP fiber samples (after subtracting out the absorbance of the alpha-chymotrypsin blanks) at 600nm as a function of incubation time. It effectively shows the absorbance of the peptides and AuNP that had gone into solution as a result of degradation by alpha-chymotrypsin. |
Latest revision as of 01:23, 27 September 2017
Project name | Main project page Previous entry Next entry |
ObjectiveToday's objective is to use a Bradford Assay to measure the absorbance of AuNP fibers that have been degraded by 1µM alpha-chymotrypsin for varying lengths of time. Protocol
DataThe above figure shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The absorbance was corrected by first averaging the raw absorbance data for each duplicate sample and then subtracting the absorbance of a Bradford blank* from the absorbance of each averaged data point at their respective wavelengths. The absorbance was then corrected by subtracting the absorbance at the isosbestic point of each sample and blank from all of the absorbance values for the respective sample and blank.
The above figure shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance was corrected in the same way that the absorbance for the alpha-chymotrypsin blanks was corrected.
The above figure shows the absorbance of the alpha-chymotrypsin blanks and the AuNP fiber samples at 600nm as a function of the amount of time that they were incubated. The absorbance values are taken directly from the absorbances in Figures 1 and 2.
The above figure shows the absorbance of the AuNP fiber samples (after subtracting out the absorbance of the alpha-chymotrypsin blanks) at 600nm as a function of incubation time. It effectively shows the absorbance of the peptides and AuNP that had gone into solution as a result of degradation by alpha-chymotrypsin.
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