User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/30: 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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# We then pipetted this mixture into a 1mL quartz cuvette and took fluorescence measurements (excitation wavelength was 390nm and emission wavelengths were 400 to 650nm). | # We then pipetted this mixture into a 1mL quartz cuvette and took fluorescence measurements (excitation wavelength was 390nm and emission wavelengths were 400 to 650nm). | ||
=== | ===Bradford Analysis of Protease Degradation=== | ||
# First, we made a stock solution of our protease, alpha-chymotrypsin, using Tris/CaCl<sub>2</sub> buffer and one of the stock masses of alpha-chymotrypsin that we measured out earlier in the semester. The concentration of this solution was 40.625µM. | # First, we made a stock solution of our protease, alpha-chymotrypsin, using Tris/CaCl<sub>2</sub> buffer and one of the stock masses of alpha-chymotrypsin that we measured out earlier in the semester. The concentration of this solution was 40.625µM. | ||
# Next, we prepared 7 samples of AuNP fibers that Dr. Hartings prepared for us: | # Next, we prepared 7 samples of AuNP fibers that Dr. Hartings prepared for us: | ||
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===Fluorescence=== | ===Fluorescence=== | ||
<center>Figure 1: Fluorescence | <center><b>Figure 1: Raw Data: Fluorescence of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)</b></center> | ||
[[Image: | [[Image:20151101 01 bonan fluorescence blanks.png|thumb|center|700px]] | ||
The above figure | The above figure is the raw data for the fluorescence of each of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. Each curve on the graph represents a different concentration of alpha-chymotrypsin. The concentrations were converted from nM to mg/mL chymotrypsin. | ||
<center>Figure 2: Fluorescence | <center><b>Figure 2: Raw Data: Fluorescence of Lysozyme Samples Digested with alpha-Chymotrypsin as a Function of the Wavelength of Incident Light (nm)</b></center> | ||
[[Image: | [[Image:20151101 01 bonan fluorescence samples.png|thumb|center|700px]] | ||
The above | The above figure is the raw data for the fluorescence of each of the lysozyme samples degraded with alpha-chymotrypsin as a function of the wavelength of incident light. Each curve on the graph represents a different concentration of lysozyme. The concentrations were converted from nM to mg/mL. | ||
<center><b>Figure 3: Fluorescence Lysozyme+Peptides as a Function of the Wavelength of Incident Light (nm)</b></center> | |||
[[Image:20151101 03 bonan fluorescence peptides.png|thumb|center|700px]] | |||
The above figure shows the fluorescence of the lysozyme+peptides in the lysozyme samples as a function of the wavelength of incident light. In order to get this data, I made the following corrections to the raw data: | |||
# I corrected the fluorescence of the lysozyme samples for instrumental noise by subtracting the fluorescence for the last .5nm measured from each of the fluorescence measurements for each respective sample | |||
# I corrected the alpha-chymotrypsin blanks for instrumental noise in the same way that I corrected the lysozyme samples | |||
# I then subtracted the corrected fluorescence values for the alpha-chymotrypsin blanks from those of the lysozyme samples for each respective wavelength and sample concentration. This resulting data was the fluorescence of just the lysozyme and peptides that were in the lysozyme sample. It effectively subtracted out any fluorescence that was from the alpha-chymotrypsin. This data is the data that the graph shows. | |||
<center><b>Figure 4: Fluorescence Intensity of Lysozyme+Peptides as a Function of the Concentration of the Lysozyme+Peptides</b></center> | |||
[[Image:20151101 04 bonan fluorescence intensity calib.png|thumb|center|700px]] | |||
The figure above will be the calibration curve for all of our fluorescence measurements of the AuNP fibers. it shows the fluorescence intensity of the lysozyme+peptides as a function of the concentration of the lysozyme+peptides. In order to get this data, I did the following: | |||
# I integrated the area under each of the curves for the corrected lysozyme fluorescence from 420-650nm | |||
# I did the same thing for the corrected alpha-chymotrypsin fluorescence | |||
# For each concentration, I subtracted integrated area of the alpha-chymotrypsin blanks from that of the lysozyme samples. This gave the fluorescence intensity of the lysozyme+peptides that were in the lysozyme samples. | |||
#I then plotted concentration vs fluorescence intensity. | |||
===Bradford Assay=== | ===Bradford Assay=== | ||
<center><b>Figure | <b><center>Figure 5: Absorbance of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)</center></b> | ||
[[Image:20151111 0930 bonan achymo 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. | |||
*NOTE: The blank was just Bradford reagent and Tris buffer. | |||
<b><center>Figure 6: Absorbance of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)</center></b> | |||
[[Image:20151111 0930 bonan aunp 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. | |||
<b><center>Figure 7: Absorbance of alpha-Chymotrypsin Blanks and AuNP Fiber Samples at 600nm as a Function of Incubation Time (min)</center></b> | |||
[[Image:20151111 0930 bonan samples blanks.png|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 5 and 6. | |||
< | <b><center>Figure 8: Absorbance of AuNP Fiber Samples-alpha-Chymotrypsin Blanks at 600nm as a Function of Incubation Time (min)</center></b> | ||
[[Image: | [[Image:20151111 0930 bonan peptides.png|thumb|center|700px]] | ||
The figure | 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. | ||
==Conclusions== | |||
===Fluorescence Assay=== | |||
The fluorescence intensity of the lysozyme increased linearly with the concentration of alpha-chymotrypsin used to digest the lysozyme. | |||
In our fluorescence assay, peptides were labeled so that they would fluoresce; as the concentration of peptide in the sample increased, the fluorescence would increase. alpha-Chymotrypsin degrades proteins, including lysozyme, which would cause the concentration of peptide in the sample to increase as the protein is degraded into peptides. Thus, since the fluorescence of the samples increased as the concentration of alpha-chymotrypsin increased, we can infer that increasing the concentration of the alpha-chymotrpysin increased the rate at which the lysozyme was degraded. | |||
===Bradford Assay=== | |||
Degrading AuNP fibers with alpha-chymotrypsin should have caused the fibers to degrade into peptides. By using a Bradford Assay, we effectively labeled peptides so that they would absorb light between 500-700nm. Thus, when we measured absorbance, the samples with more peptide would have a greater absorbance than the samples with less peptide. | |||
Theoretically, the samples with more peptide should have been the samples that had incubated longer and thus had been able to digest more AuNP fibers. By making samples that had incubated for varying lengths of time and then measuring their absorbance, our goal was to determine the rate at which our protease degraded the AuNP fibers. | |||
Unfortunately, our calibration curve does not provide much useful data about the absorbance of our samples as a function of time. We cannot really determine how fast the alpha-chymotrypsin degrades the AuNP fibers based on our Bradford Assay data. | |||
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Latest revision as of 01:13, 27 September 2017
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ObjectiveThe purpose of today's lab work is to:
ProtocolDr. Hartings's protocol for today is here. We are following his protocols for fluorescence and for the Bradford Assay. Fluorescence Analysis of Protease DegradationYesterday, we measured the fluorescence of all of our samples of alpha-chymotrypsin + lysozyme. Today, we finished the fluorescence measurements for the alpha-chymotrypsin blanks as follows:
Bradford Analysis of Protease Degradation
Data and AnalysisFluorescenceThe above figure is the raw data for the fluorescence of each of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. Each curve on the graph represents a different concentration of alpha-chymotrypsin. The concentrations were converted from nM to mg/mL chymotrypsin. The above figure is the raw data for the fluorescence of each of the lysozyme samples degraded with alpha-chymotrypsin as a function of the wavelength of incident light. Each curve on the graph represents a different concentration of lysozyme. The concentrations were converted from nM to mg/mL. The above figure shows the fluorescence of the lysozyme+peptides in the lysozyme samples as a function of the wavelength of incident light. In order to get this data, I made the following corrections to the raw data:
The figure above will be the calibration curve for all of our fluorescence measurements of the AuNP fibers. it shows the fluorescence intensity of the lysozyme+peptides as a function of the concentration of the lysozyme+peptides. In order to get this data, I did the following:
Bradford AssayThe 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* 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 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 5 and 6.
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. ConclusionsFluorescence AssayThe fluorescence intensity of the lysozyme increased linearly with the concentration of alpha-chymotrypsin used to digest the lysozyme.
Bradford AssayDegrading AuNP fibers with alpha-chymotrypsin should have caused the fibers to degrade into peptides. By using a Bradford Assay, we effectively labeled peptides so that they would absorb light between 500-700nm. Thus, when we measured absorbance, the samples with more peptide would have a greater absorbance than the samples with less peptide.
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