User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/21

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Tasks for Oct. 21

  • To prepare new dialysis of 30:1 colloid solution vs CaCl2 using MWCO 3500
  • To prepare iodide ion selective electrodes
  • To determine the molecular weight of the gel from electrophoresis started on Oct. 15

I- ISE preparation

Prodecure as describer by Dr. Fox on Oct. 21

    • Prepare 10 mL 3 M NaCl
      • Carefully remove yellow plastic covering on Ag/AgCl electrode (as instructed)
      • Place electrode in 15 mL falcon tube with enough NaCl solution to match volume in electrode
      • cover and store upright until needed
    • Prepare 50 mL 4 M KNO3
      • Add KNO3 to outer double junction cell, filling narrow tube + ~ 0.5 inches further
      • Place electrode in small vial with enough KNO3 solution to cover the vycor tip
    • Prepare 50 mL 0.01 M KI + 0.1 M KNO3
      • Cut a short piece (~ 3 in) of silver wire
      • Place in KI/KNO3 solution
      • Electrolyze the solution using the power supply
      • The standard reduction potential for Ag/AgI is -0.15 V
      • Oxidation occurs at the anode, which is connected to the red lead
      • Electrolyze at low power (low current) until the wire appears coated
    • Test your electrodes with a range of KI solutions
      • The Ag/AgCl electrode slides inside of the KNO3 bridge solution (this is a double junction electrode)
      • The Ag/AgI wire can be connected directly to a multimeter using alligator clips
      • You will continue to connect your Ag wire to the red lead on the multimeter

I- ISE

KI concentration mV measurement (mV)
2mM -198.3
5 mM -217.3
10 mM -237.0
25 mM -247.4
50mM -278.0

Image:I CC.png

30:1 colloid solution vs CaCl2 (MWCO 3500) Dialysis Data

Bradford Analysis

  • Only running bradford analysis of protein-containing solutions, that is Colloid 1, 2, 3, 4 and 5, since the protein cannot diffuse through 3500 MWCO.
  • Remove 100 μL of solution from each chamber (7 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
    • Recall, 100 μL solution + 200 μL diluted Bradford + 700 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm

Image:Bradford Colloid 221014.png
Note: The colloid solution at 50 mM CaCl2 was clear at the top, and precipitated colloid was sank in the bottom of the well. Because only the clear solution was extracted from the well, the protein detected using Bradford analysis corresponds to free lysozyme protein in solution.
Ca2+ ISE

CaCl2 concentration mV measurement (mV)
5μM (1) 26.2
Colloid (1) 14.8
50μM 24.5
Colloid (2) 30.8
500μM 59.3
Colloid (3) 45.8
5mM 70.6
Colloid (4) 66.9
50mM 86.8
Colloid (5) 85.2

UV-Vis measurements

  • No peak at 280 nm for the dialysed colloid solutions because lysozyme contains tryptophan, tyrosine, phenylalanine which are reducing agents for gold. No free protein in solution was found.

Fluorescence measurements

  • There was no fluorescence since there was no free protein in solution
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