User:Michael S. Bible/Notebook/571/2014/09/16

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Dialysis of Colloid Sample

Protocols for today were adopted from Dr. Fox's Notebook.

Description

  • Today we dialyzed a 35:1 Au:Protein solution (colloid solution) made earlier and stored in our lab drawer.
  • For dialysis, we needed to create 1 L of a 50 mM Glycine buffer, with a pH ~3.5.
    • To do this, 4 mL of 1 M HCl and 3.7608 grams of glycine were combined in a 1 L volumetric flask.
  • We also needed to prepare 500 mL of a 50 mM NaCl solution.
    • To do this, 1.46307 g of NaCl was added to water in a 500 mL volumetric flask.
  • Additionally, 25 mL of 1 g/L Lysozyme was prepared.
    • To do this, 0.025 g of Lysozyme was combined with water in a 25 mL volumetric flask.
  • We then performed dialysis using the protocol described in the "Dialysis Protocol" section below to prepare the dialysis tubes.
    • Dialysis was performed by adding 4 mL of the lysozyme solution to:
      • A 3500 g/mol MWCO tube in NaCl soak.
      • A 3500 g/mol MWCO tube in Glycine Buffer soak.
      • A 3500 g/mol MWCO tube in HPLC water soak.
      • A 25000 g/mol MWCO tube in Glycine Buffer soak.
    • An additional dialysis was performed using 4 mL of the colloid solution in:
      • A 3500 g/mol MWCO tube in Glycine Buffer soak.

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 on shaker on low speed to help prevent gradients during dialysis