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

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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

Bradford of Lys vs CaCl2.jpg

3. 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

Fluorescence Lys vs CaCl2.jpg

UV Lys vs CaCl2.jpg

NOTE Both the absorption spectra and emission spectra are indicative that an equilibrium is reached with 42 μM Lysozyme and concentrations below 5 mM CaCl2. The protein concentration reaches at max absorbance at 500 μM CaCl2 and then decreases at 5 mM and 50 mM, respectively, indicating that lysozyme is precipitating out of the solution.


6. Solution Preparation

  • Prepare new 0.6 g/L Lysozyme
    • 0.0380 g of lysozyme dissolved in 50 mL H2O
  • Prepare 5 mM Stock HCl solution
    • Add 0.5 mL of 0.5 M HCL to 50 mL distilled H2O
      • Dilutions were prepared from 5 mM HCl stock solution
        • 75 μM, 100 μM, 250 μM, and 500 μM


7. Prepare New Dialysis

  • 42 μM Lysozyme (0.6 g/L) vs HCl
    • Use 20,000 MWCO tubing
    • Add 1 mL 0.6 g/L Lysozyme to 5 cells on one side of chamber
    • Add 1 mL 75 μM, 100 μM, 250 μM, 500 μM, and 5 mM HCl to the opposite side of the chamber (one concentration per cell)
    • Secure wells by screwing them to prevent any evaporation
    • Place on low speed shaker for one week and prepare for analysis