User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/01

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Tasks for October 1

  • Analysis of Dialysis Solutions from Sept. 30

Analysis of Dialysis Solution

Procedure followed as detailed by Dr. Fox on Oct.1

  • Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:3 with 50mM Tris/50mM NaCl
    • Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm
    • Don't forget to run a blank with just Bradford & buffer
    • Don't forget to run your undialyzed Lysozyme stock
  • Transfer remainder of each cell into 20 mL extraction vials
  • Measure Ca2+ using ISE
Substance mV measurement (mV)
50mM CaCl2 (4) 80.8
Lysozyme (4) 80.3
500μM CaCl2 (3) 28.8
Lysozyme (3) 28.1
  • Transfer 100 μL to a small volume UV cuvette & measure UV absorption
    • Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
    • Measure entire 200 - 800 nm range
  • Transfer 100 μL to a small volume fluorescence cuvette & measure fluorescence
    • Be sure your cuvette is clean before hand. Use SDS, HCl, HPLC, & methanol washes
    • excitation at 280 nm
  • Transfer 500 - 700 μL to a 15 mL Falcon tube. Add 2 - 3 times volume of water & measure pH
    • You want to add minimum amount of water
    • You typically need 1.6 - 2 mL in falcon tube to cover pH probe
    • So, if you have only 500 μL left, you'll need a 4x dilution (adding 1.5 mL water)
    • Or, if you have 700 μL left, you only need a 3x dilution (adding 1.4 mL water)

Preparation of new dialysis

A new dialysis experiment was prepared using 22,000 MWCO tubing instead of 3500MWCO. <br.> The wells were matched up in the following way. Note that 1 mL of the following were added to each well:

  1. 50 mM CaCl2. Opposite to it Colloid 1
  2. 500 μM CaCl2. Opposite to it Colloid 2
  3. HPLC water. Opposite to it Lysozyme 3
  4. 0.25 mM HCl. Opposite to it Lysozyme 4
  5. 50 mM NaCl. Opposite to it Lysozyme 5
  • Inserted screws to prevent evaporation
  • Placed on low speed shaker for 1 week