User:Benjamin Friedel/Notebook/CHEM 471/2015/10/06: 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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'''Protease Preparation''' | '''Protease Preparation''' | ||
# 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM phosphate buffer (pH=8) to dry protease for a final concentration of | # 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM phosphate buffer (pH=8) to dry protease for a final concentration of 47.226µM. | ||
## Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 1uM and final tube volume of 1mL was determined | ## Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 1uM and final tube volume of 1mL was determined | ||
### V1= . | ### V1= 21.2uL | ||
# Each sample and blank tube for incubation requires a total volume of 1mL, thus 1mLtotal - 21.2uL protease = 978.8uL phosphate buffer. | |||
# Each sample and blank tube for incubation requires a total volume of 1mL, thus 1mLtotal - | |||
'''Incubation Tube Preparation (Samples and Blanks)''' | '''Incubation Tube Preparation (Samples and Blanks)''' | ||
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### 140uL of Assay Buffer | ### 140uL of Assay Buffer | ||
### 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- | ### Fluorescence was then immediately measured with a excitation wavelength of 390nm with a recorded emission spectra from 400-649.5nm. | ||
==Data== | ==Data== |
Latest revision as of 01:20, 27 September 2017
Project name | Main project page Previous entry Next entry |
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. |