User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/03/04: 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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== | ==1 μM Chymotrypsin Kinetics== | ||
* | |||
'''Protocol''' | |||
Switching to Chymotrypsin kinetics. Same protocol for OceanOptics, scan every 30 seconds for 2 hours at 37°C, spinning at 1000 RPM. | |||
For the solution prep, 104 μL of concentrated Chymotrypsin stock (24.00 μM) was diluted by 2.896 mL of Tris-10mM CaCl<sub>2</sub> buffer. | |||
'''Results''' | |||
[[Image:1uM_Chymotrypsin_Absvstime_Mar_4_Chart.png|500px]] | |||
[[Image:1uM_Chymotrypsin_Kinetics_Mar_4_Chart.png|500px]] | |||
=='''Thermolysin Reaction Preparation'''== | |||
0.00063 g of Chymotrypsin was dissolved in 1 mL of Tris/CaCl<sub>2</sub> buffer ---> stock [thermolysin] = 18.21 μM | |||
'''''Bradford/Gel Analysis samples''''' to be run: | |||
1. 0.274(6) mL of stock solution in 4.725 mL of Tris/CaCl<sub>2</sub> buffer | |||
*[thermolysin]<sub>final</sub> = 1 μM | |||
2. 0.027(4) mL of stock solution in 4.973 mL of Tris/CaCl<sub>2</sub> buffer | |||
*[thermolysin]<sub>final</sub> = 100 nM | |||
3. 0.002(7) mL of stock solution in 4.997 mL of Tris/CaCl<sub>2</sub> buffer | |||
*[thermolysin]<sub>final</sub> = 10 nM | |||
4. 10 μL of stock solution in 990 μL of Tris/CaCl<sub>2</sub> buffer --> [trypsin] = 0.1821 μM | |||
**Take 0.027(4) mL of dilution in 4.973 mL of Tris/NaCl CaCl<sub>2</sub>buffer | |||
*[thermolysin]<sub>final</sub> = 1 nM | |||
Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted. 5 mL of protease solution was added to each sample tube. A 500 μL aliquot was obtained for all protease concentrations every 5 minutes until 30 minutes, timepoints including: 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min. A 500 μL aliquot was obtained every 30 minutes after the first 30 min until 2 hours of total reaction, timepoints including: 60 min, 90 min, 120 min. The samples were shaken at 236 rpm and 37 C for the duration of the reaction time. | |||
=='''Lysozyme control digestion'''== | |||
55.6 μM lysozyme stock solution was prepared from 0.079 g of lysozyme in 100 mL of water. | |||
0.5 mL of this stock solution was combined with 4.5 mL of water in order to produce 2 - 5 mL samples of 5.56 μM lysozyme solution. These were digested with thermolysin protease with total solution protease concentrations of 1 and 100 nM. See above procedure for thermolysin stock volumes resulting in given concentrations. | |||
Samples were mistakenly pulled at the same timepoints as the thermolysin/AuNP fiber reactions, but only the 120 min timepoint will be run on the gels. | |||
Latest revision as of 00:48, 27 September 2017
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1 μM Chymotrypsin KineticsProtocol Switching to Chymotrypsin kinetics. Same protocol for OceanOptics, scan every 30 seconds for 2 hours at 37°C, spinning at 1000 RPM. For the solution prep, 104 μL of concentrated Chymotrypsin stock (24.00 μM) was diluted by 2.896 mL of Tris-10mM CaCl2 buffer. Results
Thermolysin Reaction Preparation0.00063 g of Chymotrypsin was dissolved in 1 mL of Tris/CaCl2 buffer ---> stock [thermolysin] = 18.21 μM Bradford/Gel Analysis samples to be run: 1. 0.274(6) mL of stock solution in 4.725 mL of Tris/CaCl2 buffer
2. 0.027(4) mL of stock solution in 4.973 mL of Tris/CaCl2 buffer
3. 0.002(7) mL of stock solution in 4.997 mL of Tris/CaCl2 buffer
4. 10 μL of stock solution in 990 μL of Tris/CaCl2 buffer --> [trypsin] = 0.1821 μM
Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted. 5 mL of protease solution was added to each sample tube. A 500 μL aliquot was obtained for all protease concentrations every 5 minutes until 30 minutes, timepoints including: 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min. A 500 μL aliquot was obtained every 30 minutes after the first 30 min until 2 hours of total reaction, timepoints including: 60 min, 90 min, 120 min. The samples were shaken at 236 rpm and 37 C for the duration of the reaction time. Lysozyme control digestion55.6 μM lysozyme stock solution was prepared from 0.079 g of lysozyme in 100 mL of water. 0.5 mL of this stock solution was combined with 4.5 mL of water in order to produce 2 - 5 mL samples of 5.56 μM lysozyme solution. These were digested with thermolysin protease with total solution protease concentrations of 1 and 100 nM. See above procedure for thermolysin stock volumes resulting in given concentrations. Samples were mistakenly pulled at the same timepoints as the thermolysin/AuNP fiber reactions, but only the 120 min timepoint will be run on the gels.
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