User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/03/24: Difference between revisions
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There were still fiber aggregates in solution when the reaction finished. No visible colorimetric change occurred. | There were still fiber aggregates in solution when the reaction finished. No visible colorimetric change occurred. | ||
=='''Proteinase K Reaction Preparation'''== | |||
0.00125 g of proteinase K was dissolved in 1 mL of Tris/NaCl buffer ---> stock [proteinase K] = 43.25 μM | |||
''Bradford/Gel Analysis samples:'' | |||
1. 0.116 mL of stock solution in 4.884 mL of Tris/CaCl<sub>2</sub> buffer | |||
*[proteinase K]<sub>final</sub> = 1 μM | |||
2. 0.012 mL of stock solution in 4.988 mL of Tris/CaCl<sub>2</sub> buffer | |||
*[proteinase K]<sub>final</sub> = 100 nM | |||
3. 0.001(2) mL of stock solution in 4.999 mL of Tris/CaCl<sub>2</sub> buffer | |||
*[proteinase K]<sub>final</sub> = 10 nM | |||
4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> take 0.012 mL of dilution in 4.988 mL of Tris/CaCl<sub>2</sub> buffer | |||
*[proteinase K]<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 and a 500 μL aliquot from each concentration at varying time points (depending on concentration) beginning with time point 0. The samples were kept in the incubator shaker (37C) at 236 rpm. | |||
Time points collected: | |||
* 1 μM: 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 min | |||
* 100 nM: 0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 min | |||
* 10 nM: 0, 30, 40, 50, 60, 70, 80, 90 min | |||
* 1 nM: 0, 30, 40, 50, 60, 70, 80, 90 min | |||
'''NOTE:''' While all 5 mL of the 1 μM and 100 nM samples were depleted, the 10 and 1 nM reactions were left in the incubator/shaker overnight in order to extract samples the next day. | |||
Revision as of 14:43, 31 March 2015
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1 nM Chymotrypsin Kinetics10.4 μL of dilute Chymotrypsin stock was diluted by 2.989 mL of buffer. Fibers and OceanOptics were prepared with the same protocol as previous runs. Results
There were still fiber aggregates in solution when the reaction finished. No visible colorimetric change occurred. Proteinase K Reaction Preparation0.00125 g of proteinase K was dissolved in 1 mL of Tris/NaCl buffer ---> stock [proteinase K] = 43.25 μM
1. 0.116 mL of stock solution in 4.884 mL of Tris/CaCl2 buffer
2. 0.012 mL of stock solution in 4.988 mL of Tris/CaCl2 buffer
3. 0.001(2) mL of stock solution in 4.999 mL of Tris/CaCl2 buffer
4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> take 0.012 mL of dilution in 4.988 mL of Tris/CaCl2 buffer
Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted. 5 mL of protease solution was added to each sample tube and a 500 μL aliquot from each concentration at varying time points (depending on concentration) beginning with time point 0. The samples were kept in the incubator shaker (37C) at 236 rpm. Time points collected:
NOTE: While all 5 mL of the 1 μM and 100 nM samples were depleted, the 10 and 1 nM reactions were left in the incubator/shaker overnight in order to extract samples the next day.
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