User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/10/06: 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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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==Objective== | ==Objective== | ||
The purpose of today's lab work was to use a Fluorescence Assay in order to determine the rate at which alpha-chymotrypsin digests AuNP fiber samples. | The purpose of today's lab work was to use a Fluorescence Assay in order to determine the rate at which 1µM alpha-chymotrypsin digests AuNP fiber samples. | ||
==Protocol== | ==Protocol== | ||
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## Combined, in a 600µL Eppindorf tube, the following: | ## Combined, in a 600µL Eppindorf tube, the following: | ||
### 20uL of the blank or sample | ### 20uL of the blank or sample | ||
### 140uL of | ### 140uL of Assay Buffer | ||
### 40uL of | ### 40uL of Assay Reagent | ||
## Measured the fluorescence with an excitation wavelength of 390nm and an emission spectrum from 400 to 600nm | ## Measured the fluorescence with an excitation wavelength of 390nm and an emission spectrum from 400 to 600nm | ||
# We then repeated steps 4-5 for the 10 min sample and blank. | # We then repeated steps 4-5 for the 10 min sample and blank. | ||
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==Data== | ==Data== | ||
<center><b>Figure 1: Fluorescence of alpha-Chymotrypsin Blanks as a Function of the Wavelength of Incident Light (nm)</b></center> | <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 1006 01 bonan fluoresc blanks.png|thumb|center|700px]] | ||
The figure above shows the fluorescence of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend | The figure above shows the raw data for the fluorescence of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend. | ||
<center><b>Figure 2: Fluorescence of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)</b></center> | <center><b>Figure 2: Raw Data: Fluorescence of AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)</b></center> | ||
[[Image: | [[Image:20151101 1006 02 bonan fluoresc samples.png|thumb|center|700px]] | ||
The figure above shows the fluorescence of the AuNP fiber samples, which incubated with alpha-chymotrypsin, as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend | The figure above shows the raw data for the fluorescence of the AuNP fiber samples, which incubated with alpha-chymotrypsin, as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend. | ||
<center><b>Figure 3: Fluorescence Intensity of the alpha-Chymotrypsin Blanks and the AuNP Fiber Samples as a Function of the Incubation Time (min)</b></center> | <center><b>Figure 3: Fluorescence Intensity of the alpha-Chymotrypsin Blanks and the AuNP Fiber Samples as a Function of the Incubation Time (min)</b></center> | ||
[[Image: | [[Image:20151101 1006 03 bonan fluoresc intensity both.png|thumb|center|700px]] | ||
The above figure shows the fluorescence intensity of both the alpha-chymotrypsin blanks (in blue) and the AuNP fiber samples (in red) as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by | The above figure shows the fluorescence intensity of both the alpha-chymotrypsin blanks (in blue) and the AuNP fiber samples (in red) as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by doing the following: | ||
# I corrected the alpha-chymotrypsin blanks for instrumental noise by subtracting the fluorescence for the last 0.5nm from all of the other fluorescence measurements for each blank | |||
# I did the same correction for instrumental noise for the AuNP fiber samples | |||
# I integrated the area under the fluorescence curve for each blank starting at 420nm. This gave the fluorescence intensity of each blank. | |||
# I integrated the area under the fluorescence curve for each sample starting at 420nm. This gave the fluorescence intensity of each sample. | |||
# I graphed both integrations | |||
Interestingly, both the blanks and the samples seemed to reach a maximum fluorescence intensity at an incubation time between 30-45 minutes. The fluorescence intensity then decreased sharply for the blanks and then began to level off. The same pattern was observed in the AuNP fiber samples, though the drop in intensity was not as steep. | |||
<center><b>Figure 4: Fluorescence Intensity of the AuNP Fibers+Peptides as a Function of the Incubation Time (min)</b></center> | |||
[[Image:20151101 1006 04 bonan fluoresc intensity peptides.png|thumb|center|700px]] | |||
The above figure shows the fluorescence intensity of the AuNP fibers+peptides as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by subtracting the fluorescence intensity of the alpha-chymotrypsin blanks from that of the AuNP fiber samples. This effectively subtracted out any fluorescence intensity in the fiber samples that would have been from the alpha-chymotrypsin, giving only the fluorescence intensity of the intact AuNP fibers and the chymotrypsin-digested AuNP fibers (peptides). | |||
<center><b>Figure 5: Concentration of AuNP Fibers+Peptide (mg/mL) as a Function of Incubation Time in alpha-Chymotrypsin (min)</b></center> | |||
[[Image:20151101 1006 05 bonan concentration.png|thumb|center|700px]] | |||
The above figure shows the concentration of the AuNP fibers+peptides as a function of the amount of time that the solutions were incubated. The concentration of the AuNP fibers+peptides was determined from the data in Figure 4: these data points were divided by the slope of the calibration curve from [[User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/30|September 30]] (figure 4), which was 475965. This converted the fluorescence intensity to the concentration of AuNP fibers+peptides. | |||
Latest revision as of 01:14, 27 September 2017
Project name | Main project page Previous entry Next entry |
ObjectiveThe purpose of today's lab work was to use a Fluorescence Assay in order to determine the rate at which 1µM alpha-chymotrypsin digests AuNP fiber samples. Protocol
DataThe figure above shows the raw data for the fluorescence of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend.
The figure above shows the raw data for the fluorescence of the AuNP fiber samples, which incubated with alpha-chymotrypsin, as a function of the wavelength of incident light. The different curves on the graph represent different incubation times, as indicated by the legend.
The above figure shows the fluorescence intensity of both the alpha-chymotrypsin blanks (in blue) and the AuNP fiber samples (in red) as a function of the amount of time that the solutions were incubated. The fluorescence intensity was determined by doing the following:
Interestingly, both the blanks and the samples seemed to reach a maximum fluorescence intensity at an incubation time between 30-45 minutes. The fluorescence intensity then decreased sharply for the blanks and then began to level off. The same pattern was observed in the AuNP fiber samples, though the drop in intensity was not as steep.
The above figure shows the concentration of the AuNP fibers+peptides as a function of the amount of time that the solutions were incubated. The concentration of the AuNP fibers+peptides was determined from the data in Figure 4: these data points were divided by the slope of the calibration curve from September 30 (figure 4), which was 475965. This converted the fluorescence intensity to the concentration of AuNP fibers+peptides.
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