User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/03: Difference between revisions
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== | ==March 3<sup>rd</sup>, 2015== | ||
==Enzyme Condition Notes== | |||
'''Proteinase K''' | |||
*MW: 28,900 Da | |||
*optimal pH: 8.0 | |||
*optimal temperature range: 20-60C | |||
*optimal concentration range: 1.73 - 3.46 uM | |||
*Ca is a stabilizer especially at low concentrations (1-5 uM) | |||
*suggested buffer: 50 mM Tris-HCl (pH 7.5) 5 mM CaCl<sub>2</sub> | |||
'''Trypsin''' | |||
*MW: 23,300 Da | |||
*optimal pH: 7.5-8.5 | |||
*optimal temperature range: | |||
*suggested buffer: 46 mM Tris-HCl (pH 8.1) 11.5 mM CaCl<sub>2</sub> | |||
'''Chymotrypsin''' | |||
*MW: 25,000 Da | |||
*optimal pH: 7.8-8.0 | |||
*100 mM Tris-HCl (pH 8.0), 10 mM CaCl<sub>2</sub> | |||
OR 80 mM Tris-HCl (pH 7.8) with 0.1 M CaCl<sub>2</sub> | |||
'''Thermolysin''' | |||
*MW: 36,200 Da | |||
*optimal pH: 8.0 | |||
*recommended enzyme:protein ratios of 1:20 - 1:50 | |||
*digestion buffer: 50 mM Tris-HCl (pH 8.0) 0.5 mM CaCl<sub>2</sub> | |||
'''Pepsin''' | |||
*MW: 34,600 Da | |||
*optimal pH: 1.0-3.0 | |||
*recommended enzyme:protein ratios of 1:20 - 1:100 | |||
=='''Pepsin Reaction Preparation'''== | =='''Pepsin Reaction Preparation'''== | ||
0.00166 g of pepsin was dissolved in 1 mL of | 0.00166 g of pepsin was dissolved in 1 mL of 5 mM citrate/0.2 M NaCl buffer ---> stock [pepsin] = 47.98 μM | ||
'''''Bradford/Gel Analysis samples''''' to be run: | |||
1. 0.104(2) mL of stock solution in 4.896 mL of citrate/NaCl buffer | |||
*[pepsin]<sub>final</sub> = 1 μM | |||
2. 0.010(4) mL of stock solution in 4.989 mL of citrate/NaCl buffer | |||
*[pepsin]<sub>final</sub> = 100 nM | |||
3. 0.001(0) mL of stock solution in 4.999 mL of citrate/NaCl buffer | |||
*[pepsin]<sub>final</sub> = 10 nM | |||
4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.4798 μM | |||
**Take 0.010(4) mL of dilution in 4.989 mL of Tris/NaCl buffer | |||
*[pepsin]<sub>final</sub> = 1 nM | |||
=='''Thermolysin Reaction Preparation'''== | =='''Thermolysin Reaction Preparation'''== | ||
0.00063 g of Chymotrypsin was dissolved in 1 mL of Tris/NaCl buffer ---> stock [thermolysin] = | 0.00063 g of Chymotrypsin was dissolved in 1 mL of Tris/NaCl 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/NaCl buffer | |||
*[thermolysin]<sub>final</sub> = 1 μM | |||
2. 0.027(4) mL of stock solution in 4.973 mL of Tris/NaCl buffer | |||
*[thermolysin]<sub>final</sub> = 100 nM | |||
3. 0.002(7) mL of stock solution in 4.997 mL of Tris/NaCl buffer | |||
*[thermolysin]<sub>final</sub> = 10 nM | |||
4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.1821 μM | |||
**Take 0.027(4) mL of dilution in 4.973 mL of Tris/NaCl buffer | |||
*[thermolysin]<sub>final</sub> = 1 nM | |||
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March 3rd, 2015Enzyme Condition NotesProteinase K
Trypsin
Chymotrypsin
OR 80 mM Tris-HCl (pH 7.8) with 0.1 M CaCl2 Thermolysin
Pepsin
Pepsin Reaction Preparation0.00166 g of pepsin was dissolved in 1 mL of 5 mM citrate/0.2 M NaCl buffer ---> stock [pepsin] = 47.98 μM Bradford/Gel Analysis samples to be run: 1. 0.104(2) mL of stock solution in 4.896 mL of citrate/NaCl buffer
2. 0.010(4) mL of stock solution in 4.989 mL of citrate/NaCl buffer
3. 0.001(0) mL of stock solution in 4.999 mL of citrate/NaCl buffer
4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.4798 μM
Thermolysin Reaction Preparation0.00063 g of Chymotrypsin was dissolved in 1 mL of Tris/NaCl 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/NaCl buffer
2. 0.027(4) mL of stock solution in 4.973 mL of Tris/NaCl buffer
3. 0.002(7) mL of stock solution in 4.997 mL of Tris/NaCl buffer
4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.1821 μM
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