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==
==Enzyme Condition Notes==


Revision as of 14:41, 1 March 2015

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March 3rd, 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 CaCl2

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 CaCl2

Chymotrypsin

  • MW: 25,000 Da
  • optimal pH: 7.8-8.0
  • 100 mM Tris-HCl (pH 8.0), 10 mM CaCl2

OR 80 mM Tris-HCl (pH 7.8) with 0.1 M CaCl2

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 CaCl2

Pepsin

  • MW: 34,600 Da
  • optimal pH: 1.0-3.0
  • recommended enzyme:protein ratios of 1:20 - 1:100

Pepsin Reaction Preparation

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]final = 1 μM

2. 0.010(4) mL of stock solution in 4.989 mL of citrate/NaCl buffer

  • [pepsin]final = 100 nM

3. 0.001(0) mL of stock solution in 4.999 mL of citrate/NaCl buffer

  • [pepsin]final = 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]final = 1 nM

Thermolysin Reaction Preparation

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]final = 1 μM

2. 0.027(4) mL of stock solution in 4.973 mL of Tris/NaCl buffer

  • [thermolysin]final = 100 nM

3. 0.002(7) mL of stock solution in 4.997 mL of Tris/NaCl buffer

  • [thermolysin]final = 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]final = 1 nM


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