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==


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*recommended enzyme:protein ratios of 1:20 - 1:100
*recommended enzyme:protein ratios of 1:20 - 1:100


=='''Pepsin Reaction Preparation'''==
most of these also talked about adding an inhibitor to stop it...
 
=='''Bradford Analysis of Trypsin Reaction Samples'''==
 
250 μL of each protein sample at every time point was combined with 200 μL of Bradford reagent (1:4 Bradford:Tris/NaCl), and 550 mL of Tris/NaCl buffer and run from 400 - 800 nm on the UV/Vis spectrophotometer. The following are the results with the blank subtracted out and zeroed at a wavelength of 535 nm.
 
* Kinetics of Chymotropsin at 1μM, 100nM, 10nM and 1nM
 
[[Image:1uM.Chymotrypsin.Kinetics.png]]
 
[[Image:100nM.Chymotrypsin.Kinetics.png]]


0.00166 g of pepsin was dissolved in 1 mL of 5 mM citrate/0.2 M NaCl buffer ---> stock [pepsin] = 47.98 μM
[[Image:10nM.Chymotrypsin.Kinetics.png]]


'''''Bradford/Gel Analysis samples''''' to be run:
[[Image:1nM.Chymotrypsin.Kinetics.png]]


1. 0.104(2) mL of stock solution in 4.896 mL of citrate/NaCl buffer
=='''1 nM Trypsin In Situ Kinetics'''==
*[pepsin]<sub>final</sub> = 1 μM


2. 0.010(4) mL of stock solution in 4.989 mL of citrate/NaCl buffer
Ran the final Trypsin In Situ reaction.  
*[pepsin]<sub>final</sub> = 100 nM


3. 0.001(0) mL of stock solution in 4.999 mL of citrate/NaCl buffer
'''Protocol'''
*[pepsin]<sub>final</sub> = 10 nM


4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.4798 μM
9.07 µL of the diluted stock was added to 2.991 mL of Tris/NaCl buffer. Fibers were spun at 300 RPM for 20 minutes (the fibers did not sediment after the first 10 minutes), and the liquid was extracted. 2 mL of the Trypsin was added to the cuvette with minimum amount of liquid from AuNP fibers. OceanOptics was run at 37°C, spinning at 1000 RPM, scanning every 5 minutes for 6 hours total.  
**Take 0.010(4) mL of dilution in 4.989 mL of Tris/NaCl buffer
*[pepsin]<sub>final</sub> = 1 nM


=='''Thermolysin Reaction Preparation'''==
'''Results'''


0.00063 g of Chymotrypsin was dissolved in 1 mL of Tris/NaCl buffer ---> stock [thermolysin] = 18.21 μM
[[Image:1nM_Trypsin_AbsvsTime_Chart.png|500px]]


'''''Bradford/Gel Analysis samples''''' to be run:
[[Image:1nM_Trypsin_Kinetics_Chart.png|500px]]


1. 0.274(6) mL of stock solution in 4.725 mL of Tris/NaCl buffer
There was barely a change in absorbance with this concentration of Trypsin.
*[thermolysin]<sub>final</sub> = 1 μM


2. 0.027(4) mL of stock solution in 4.973 mL of Tris/NaCl buffer
=='''CaCl<sub>2</sub> Buffer Preparation'''==
*[thermolysin]<sub>final</sub> = 100 nM


3. 0.002(7) mL of stock solution in 4.997 mL of Tris/NaCl buffer
A 50 mM Tris-HCl/ 10 mM CaCl<sub>2</sub> pH 8.155 buffer was prepared with 6.05923 g of Tris-HCl and 1.1098 g of CaCl<sub>2</sub> in a 1000 mL volumetric flask. Acid was added to a final pH of 8.155, however N/2 HCl acid contributed to a higher overall volume of 1040 mL.
*[thermolysin]<sub>final</sub> = 10 nM


4. 10 μL of stock solution in 990 μL of Tris/NaCl buffer --> [trypsin] = 0.1821 μM
Final concentrations:
**Take 0.027(4) mL of dilution in 4.973 mL of Tris/NaCl buffer
 
*[thermolysin]<sub>final</sub> = 1 nM
[Tris-HCl]<sub>final</sub> = 48 mM
[CaCl<sub>2</sub>]<sub>final</sub> = 9.6 mM


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Latest revision as of 00:47, 27 September 2017

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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

most of these also talked about adding an inhibitor to stop it...

Bradford Analysis of Trypsin Reaction Samples

250 μL of each protein sample at every time point was combined with 200 μL of Bradford reagent (1:4 Bradford:Tris/NaCl), and 550 mL of Tris/NaCl buffer and run from 400 - 800 nm on the UV/Vis spectrophotometer. The following are the results with the blank subtracted out and zeroed at a wavelength of 535 nm.

  • Kinetics of Chymotropsin at 1μM, 100nM, 10nM and 1nM

1 nM Trypsin In Situ Kinetics

Ran the final Trypsin In Situ reaction.

Protocol

9.07 µL of the diluted stock was added to 2.991 mL of Tris/NaCl buffer. Fibers were spun at 300 RPM for 20 minutes (the fibers did not sediment after the first 10 minutes), and the liquid was extracted. 2 mL of the Trypsin was added to the cuvette with minimum amount of liquid from AuNP fibers. OceanOptics was run at 37°C, spinning at 1000 RPM, scanning every 5 minutes for 6 hours total.

Results

There was barely a change in absorbance with this concentration of Trypsin.

CaCl2 Buffer Preparation

A 50 mM Tris-HCl/ 10 mM CaCl2 pH 8.155 buffer was prepared with 6.05923 g of Tris-HCl and 1.1098 g of CaCl2 in a 1000 mL volumetric flask. Acid was added to a final pH of 8.155, however N/2 HCl acid contributed to a higher overall volume of 1040 mL.

Final concentrations:

[Tris-HCl]final = 48 mM [CaCl2]final = 9.6 mM


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