User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/03: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 47: Line 47:


most of these also talked about adding an inhibitor to stop it...
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]]
[[Image:10nM.Chymotrypsin.Kinetics.png]]
[[Image:1nM.Chymotrypsin.Kinetics.png]]




Revision as of 13:09, 4 March 2015

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Monika_Gasiorek/Notebook/CHEM-571_2014F/2015/03/03&oldid=870486"