User:Khyra A. Neal/Notebook/Chem 571/2014/10/29: Difference between revisions

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* Use SDS, HCl, HPLC, & methanol to clean cuvette after each use
* Use SDS, HCl, HPLC, & methanol to clean cuvette after each use
* Fluorescence spectra was obtained to determine how binding of the protein was affected
* Fluorescence spectra was obtained to determine how binding of the protein was affected
[[Image:Fluorescence_Lys_vs_CaCl2.jpg]]






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Revision as of 18:33, 30 October 2014

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October 29, 2014

Tasklist

1. Extract Lysozyme and CaCl2 solutions

  • Solutions were prepared on October 28, 2014
  • Extracted with a pasteur pipette and transferred to 30 mL extraction vials

2. Bradford Analysis

  • Extracted Lysozyme solutions that were dialyzed against CaCl2
  • 200 μL Bradford Reagent (diluted 1:3)
  • 50 μL Sample Solution
  • 750 μL 50 mM Tris/ 50 mM NaCl Buffer
  • Run UV-Vis between 400 nm and 800 nm

3. Ca2+ ISE

Ca2+ ISE

CaCl2 concentration mV
5μM CaCl2 11.8
Lys (1 ) 4.6
50μM CaCl2 4.2
Lys (2) 0.1
500μM CaCl2 27.4
Lys (3) 24.4
5mM CaCl2 52.1
Lys (4) 51.3
50mM CaCl2 77.3
Lys (5) 77.0

5. UV-Vis and Fluorescence

  • Sample Solutions were diluted 1:100
  • Transfer 100 μL of sample solution to a small volume cuvette
  • Measure absorbance between 200 nm and 800 nm
  • Use SDS, HCl, HPLC, & methanol to clean cuvette after each use
  • Fluorescence spectra was obtained to determine how binding of the protein was affected



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