User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/10/29

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Tasks for October 29

  • Dialysis of 0.6 g/L Lysozyme vs HCl using MWCO 20000

Preparation of HCl stock solutions

  • Need to prepare HCl stock solution of 75 μM, 100 μM, 250 μM, 500 μM and 5 mM with distilled water.
    • The initial stock solution of 5 mM HCl was prepared by diluting 0.5 mL of 0.5 N HCl to 50 mL using distilled water
      • 0.5v1=5×10-3(50mL); v1=0.5 mL.
    • Made remaining stock solutions from 5 mM stock

Preparation of new 0.6g/L lysozyme stock

  • Had to prepare new 0.6 g/L lysozyme since it precipitated out of solution.
  • Made 50 mL of 0.6 g/L lysozyme solution by adding 0.0380 g of lysozyme (Theoretical value 0.030 g, but some may have been lost in transfer).

Dialysis of 0.6 g/L Lysozyme vs HCl using MWCO 20000

  • Note: 100 μM HCl completed evaporated from well, despite being screwed shut.

Bradford Analysis

  • Ran bradford analysis of all protein-containing solutions, that is Lysozyme 1, 2, 3, 4 and 5, and all the HCl solutions.
  • 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

pH measurements

Substance pH measurement
75 μM HCl (1) 7.142
Lys 1 7.116
Lys 2 7.423
250 μM HCl (3) 5.707
Lys 3 6.158
500μM HCl (4) 4.944
Lys 4 4.921
5mM HCl (5) 2.996
Lys 5 3.025