User:Benjamin Friedel/Notebook/CHEM 471/2015/10/06: Difference between revisions
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### 40uL of Assay Reagent | ### 40uL of Assay Reagent | ||
### Fluorescence was then immediately measured with a excitation wavelength of 390nm with a recorded emission spectra from 400-648.5nm. | ### Fluorescence was then immediately measured with a excitation wavelength of 390nm with a recorded emission spectra from 400-648.5nm. | ||
==Data== | |||
Figure 1: [[Image:JNB.10.6.15.1uM.sampleandblank.png|thumb|center|700px]] | |||
Figure 1 above shows end wavelength corrected fluorescence intensity of both the alpha-chymotrypsin incubated fiber samples and blanks. To correct for instrumental noise, the fluorescence intensity for the last .5nm was subtracted from all fluorescence measurements for each specific blank and sample. Then the area under each fluorescence curve (samples and blanks) was integrated starting at 420 nm and ranging to 649.5nm. Then integrated fluorescence intensity values were plotted for samples and blanks separately. | |||
Figure 2: [[Image:JNB.10.6.15.1uM.correctedsamplesintensity.png|thumb|center|700px]] | |||
Figure 2 above shows blank corrected integrated fluorescence intensity over incubation time. The integrated fluorescence intensity of the blanks was subtracted from those of the samples to correct for the blanks. Subtracting our blank fluorescence accounted for fluorescence from the protease (alpha-chymotrypsin), thus this graph shows the fluorescence intensity of AuNPs and peptide fragments resulting from the digest of AuNPs. | |||
Figure 3: [[Image:JNB.10.6.15.1uM.correctedsamplesconcentration.png|thumb|center|700px]] | |||
Figure 3 above shows the calibration curve derived concentration of AuNP fibers in solution in addition to peptide fragments over protease incubation time. Concentrations were derived from inputting blank and instrumental noise corrected integrated fluorescence values into the calibration curve derived equation for lysozyme from [[User:Benjamin Friedel/Notebook/CHEM 471/2015/09/29]]. The equation was y=475965x with y being the fluorescence intensity and x being the concentration. By inputting measured, corrected fluorescence intensities, concentrations were calculated for each incubation time point. | |||
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__NOTOC__ | __NOTOC__ |
Revision as of 09:58, 22 November 2015
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ObjectiveThe objective for today is to measure the fluorescence of lysozyme AuNP fibers over various incubation times with alpha chymotrypsin. This will allow us to calculate the concentration of AuNP Fibers and Peptides in solution based on a calibration curve. ProtocolProtease Preparation
Incubation Tube Preparation (Samples and Blanks)
Fluorescence Assay Preparation
DataFigure 1:Figure 1 above shows end wavelength corrected fluorescence intensity of both the alpha-chymotrypsin incubated fiber samples and blanks. To correct for instrumental noise, the fluorescence intensity for the last .5nm was subtracted from all fluorescence measurements for each specific blank and sample. Then the area under each fluorescence curve (samples and blanks) was integrated starting at 420 nm and ranging to 649.5nm. Then integrated fluorescence intensity values were plotted for samples and blanks separately.
Figure 3 above shows the calibration curve derived concentration of AuNP fibers in solution in addition to peptide fragments over protease incubation time. Concentrations were derived from inputting blank and instrumental noise corrected integrated fluorescence values into the calibration curve derived equation for lysozyme from User:Benjamin Friedel/Notebook/CHEM 471/2015/09/29. The equation was y=475965x with y being the fluorescence intensity and x being the concentration. By inputting measured, corrected fluorescence intensities, concentrations were calculated for each incubation time point. |