User:Benjamin Friedel/Notebook/CHEM 471/2015/10/28: Difference between revisions
(Autocreate 2015/10/28 Entry for User:Benjamin_Friedel/Notebook/CHEM_471) |
No edit summary |
||
Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### 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== | |||
Ocean Optics Spectrophotometry was used to measure absorbance of an AuNP fibers sample over time as it was incubated with the protease, alpha-chymotrypsin. This was performed to track how alpha-chymotrypsin digests lysozyme based AuNP fibers. | |||
==Protocol== | |||
'''AuNP Fiber Sample Preparation''' | |||
# AuNP Fiber samples (4) synthesized by Dr. Hartings were centrifuged at 300rpm for 10min. Afterwards, the supernatant was gently removed as to not disturb the fibers and release them into solution. | |||
## The samples were then resuspended and combined (gently) in 1 mL of 50mMTris/ 20mM CaCl2 Buffer(pH=8) | |||
'''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 42.188µM. | |||
## Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 100nM and final tube volume of 3mL was determined | |||
### V1= 4.74uL | |||
# Each sample and blank tube for incubation requires a total volume of 1mL, thus 3mLtotal - 4.74uL protease - 1mL resuspended AuNP fibers = 1995.26uL Tris/CaCl buffer. | |||
'''Incubation Mix Preparation''' | |||
# To the Ocean Optics Cuvette, the 1mL total resuspended fiber samples was added as well as the volume of phosphate buffer as shown above. | |||
## Experimental and save parameters were set to leave 2min between scans and to only scan for 2.5 hours. Additionally, the scan ranged from 190.96nm-891.81nm. | |||
## To begin the experiment, the lower save file button and then the play button were clicked, creating an immediate first save file. | |||
# Then, immediately after the second save file was generated after 2min, the volume of protease (alpha chymotrypsin) as shown above was added to the sample. | |||
==Data== | |||
'''Data Processing''' | |||
In this experiment, the blank or control data set is considered to be the first data point collected after the initial data point collection. To correct for this blank data (without protease), the absorbance values at each wavelength were subtracted from the absorbance data for each 2 minute measurement after addition of protease (alpha chymotrypsin). Then, to correct for instrumental noise, the absorbance values for the last 60nm for each 2min scan were averaged individually and subtracted from that individual scan. | |||
Figure 1 [[Image:JNB.10.28.15.OO.100nM.png|thumb|center|700px]] | |||
Figure 1 above shows the absorbance values for each scan after the first 2min at 530nm are shown as time progressed. This is because AuNP fibers were previously determined to have an absorbance max at 530nm, so their breakdown into solution upon digestion of AuNP fibers by alpha-chymotrypsin was monitored. This figure, in contrast to data collected on 10/07/15 shows that before the protease was added, lower absorbance. After protease addition absorbance increases and plateau's around A=.11. | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Revision as of 11:43, 22 November 2015
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
ObjectiveOcean Optics Spectrophotometry was used to measure absorbance of an AuNP fibers sample over time as it was incubated with the protease, alpha-chymotrypsin. This was performed to track how alpha-chymotrypsin digests lysozyme based AuNP fibers. ProtocolAuNP Fiber Sample Preparation
Protease Preparation
Incubation Mix Preparation
DataData Processing In this experiment, the blank or control data set is considered to be the first data point collected after the initial data point collection. To correct for this blank data (without protease), the absorbance values at each wavelength were subtracted from the absorbance data for each 2 minute measurement after addition of protease (alpha chymotrypsin). Then, to correct for instrumental noise, the absorbance values for the last 60nm for each 2min scan were averaged individually and subtracted from that individual scan.
Figure 1 above shows the absorbance values for each scan after the first 2min at 530nm are shown as time progressed. This is because AuNP fibers were previously determined to have an absorbance max at 530nm, so their breakdown into solution upon digestion of AuNP fibers by alpha-chymotrypsin was monitored. This figure, in contrast to data collected on 10/07/15 shows that before the protease was added, lower absorbance. After protease addition absorbance increases and plateau's around A=.11. |