User:Douglas M. Fox/Notebook/AU CHEM-571 F2011 Lab Support/2014/09/16

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  • If your AuNP colloid solution has precipitated, remake a 30:1 Au:protein (or lower Au) solution
  • Prepare 500 mL 50 mM NaCl
  • Prepare 1 L 50 mM Glycine buffer, pH 3.5
    • As shown in prior day notebook page, I used 0.4 mL of 1 M HCl to make 100 mL of buffer
  • Prepare 25 mL 1 g/L Lysozyme
  • Dialyze about 4 mL of lysozyme in about 3 in of dialysis tube
    • Prepare 1 3500 g/mol MWCO tubing for NaCl, Glycine, & pure water soak
    • Prepare 1 15k g/mol MWCO tubing for Glycine soak
    • Prepare 1 25k g/mol MWCO tubing for Glycine soak
  • Dialyze 4 mL of your colloid solution in 3500 g/mol MWCO tubing for Glycine soak
  • Construct pH and conductivity calibration curve for both NaCl and Glycine buffer
    • For each solution, prepare 2x, 5x, 10x, 20x, 50x, 100x, 200x, and 500x dilutions
    • Why don't we need anything lower than 100 μM?
    • You will need 12 - 15 mL of each solution. Consider serial dilutions for each order of magnitude.
  • Prepare 12 - 15 mL of 0.25 μM HCl from your HCl stock solution
  • Measure the pH and conductivity of this dilution
  • Use multi-well dialysis apparatus to dialyze lysozyme against:
    • 50 μM, 25 μM, 2.5 μM, and 0.25 μM glycine buffer
    • fill fifth well with 0.25 μM HCl
    • before loading wells, you will wet a dialysis sheet and clamp it between the plastic plates
    • fill one side with 1 mL of lysozyme and the other side with 1 mL of your buffer
  • Analyze inside and outside dialysis membranes (pH, conductivity, Bradford) tomorrow while you wait for electrophoresis

Dialysis Protocol

  1. Cut about a 3" length of dialysis tubing
    • 25,000 MWCO tubing is stored in an azide solution to prevent mold (use care; NaN3 is toxic)
    • keep the 25,000 MWCO tubing wet to prevent pore shrinkage
  2. wash cut tubing with DI water, both inside and out
    • 3,500 MWCO and 15,000 MWCO are dry and need to be wet prior to filling
    • 25,000 MWCO needs to be rinsed to remove the sodium azide
  3. flatten tubing and remove as much residual water as possible with gloves fingers
  4. clamp one end 1/4 - 1/2" from edge
  5. open other end and transfer your measured protein solution inside
  6. carefully flatten open end and clamp, making sure no liquid escapes
  7. rinse with DI water, particularly the ends, which may have residual protein solution on it
  8. place into a 150 mL beaker (or 250 mL or 400 mL)
  9. measure out 100 - 200 mL of your dialyzing solution
  10. label beaker, cover with parafilm, and leave in your drawer overnight
    • we may use the shaker on low speed to help prevent gradients during dialysis
    • you should have 8 solutions when you are finished