User:Beatriz Gimenez De C./Notebook/571/2014/10/01

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Tasks

  • Remove the 10 20 μL of solutions from each chamber and run Bradford analysis
    • Dilute Bradford reagent to 1:3 with 50mM Tris/50mM NaCl
      • 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
    • Measure absorbance in 400 - 800 nm
      • run a blank with just Bradford & buffer
      • run undialyzed Lysozyme stock
  • Transfer remainder of each cell into 20 mL extraction vials
  • Measure Ca2+ using ISE
  • Transfer 100 μL to a small volume UV cuvette & measure UV absorption
    • clean cuvette before hand using SDS, HCl, HPLC, & methanol washes
    • Measure entire 200 - 800 nm range
  • Transfer 100 μL to a small volume fluorescence cuvette & measure fluorescence
    • Clean cuvette before hand using 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
    • add minimum amount of water
    • Use 1.6 - 2 mL in falcon tube with pH probe
    • Use 4x dilution of the 500 μL left of solution (adding 1.5 mL water)

Results

  • Ca2+ ISE measurments
Substance mesurments (mV)
50mM CaCl2 (4) 80.8
Lysozyme (4) 80.3
500μM CaCl2 (3) 28.8
Lysozyme (3) 28.1
  • Bradford data

10-01-2014 Dialysis Bradford data.png