User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/30: Difference between revisions
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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. | 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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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:
Brasford Analysis of Protease Degradation
Data and AnalysisFluorescenceThe above figure shows the fluorescence of lysozyme in samples of varying concentrations of alpha-chymotrypsin as a function of the wavelength of incident light. The fluorescence of each sample was corrected for the blanks by subtracting the fluorescence of the blank from the fluorescence of the sample for every wavelength measured. Each curve on the graph represents a different concentration of alpha-chymotrypsin. The above image shows the fluorescence intensity of lysozyme as a function of the concentration of alpha-chymotrypsin that it is reacted with. Bradford AssayThe figure above shows the absorbance of the blank as a function of the wavelength of incident light. The blank is the same blank that was made and measured in the protocol from September 23. This blank consisted of the Bradford Assay Reagent, Bradford Assay Buffer, and 50mM Tris + 50mM NaCl buffer. There was no protease, protein, or AuNP in this blank.
The figure above shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. Each colored curve represents a blank that was incubated for a specific amount of time as indicated in the legend. These values are not corrected; they are the raw data.
The figure above shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. Each colored curve represents a sample that was incubated for a specific amount of time as indicated in the legend. These values are not corrected; they are the raw data.
The figure above shows the absorbance of the alpha-chymotrypsin blanks as a function of the wavelength of incident light. The absorbance values for each of the alpha-chymotrypsin blanks were corrected by subtracting the absorbance value of the blank from the absorbance value of the alpha-chymotrypsin blanks for every wavelength. Each colored curve represents an alpha-chymotrypsin blank that was incubated for a specific amount of time as indicated in the legend.
The figure above shows the absorbance of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance values for each of the samples were corrected by subtracting the absorbance value of the blank from the absorbance value of the samples for every wavelength. Each colored curve represents a sample that was incubated for a specific amount of time as indicated in the legend.
The figure above shows the absorbance of each of the alpha-chympotrypsin blanks as a function of the wavelength of incident light. The absorbance was first corrected for the blank by subtracting the absorbance of the blank at each wavelength from the absorbance of the alpha-chymotrypsin blank at each wavelength. This procedure was done for every alpha-chymotrypsin blank. The absorbance was then corrected for the isosbestic point. Since the isosbestic point was at 535nm, and the absorbance at 535nm was not 0 after correcting for the blank, the absorbance was corrected again in order to make the isosbestic point have an absorbance of 0. Thus, the absorbance of each alpha-chymotrypsin blank at 535nm was subtracted from every absorbance measurement for that alpha-chymotrypsin blank.
The figure above shows the absorbance of each of the AuNP fiber samples as a function of the wavelength of incident light. The absorbance was first corrected for the blank by subtracting the absorbance of the blank at each wavelength from the absorbance of the sample at each wavelength. This procedure was done for every sample. The absorbance was then corrected for the isosbestic point in the same way that it was corrected for the alpha-chymotrypsin blanks (see Figure 8).
The figure above is the calibration curve for both the alpha-chymotrypsin blanks and the AuNP fiber samples. It shows the absorbance of the blanks and samples as a function of incubation time at a wavelength of incident light of 600nm.
ConclusionsFluorescence AssayBradford 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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