User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/14

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Purpose

  • Perform dialysis on purified ADA solution to decrease the concentration of salt in ADA solutions.
  • Make Au/ADA and Au/HRP samples with ratio between 60 and 150 in 10 increments

Procedure

  • Dialysis of ADA solution was performed in the following way:
    • 4000mL of distilled water was placed in a large 5000mL dialysis beaker.
    • For ADA solution purified in 10/03/2012, about 10cm of SnakeSkin Dialysis Tubing from Thermo Scientific with a diameter 22mm was used. The bottom of dialysis tubing was folded twice over and clipped with a dialysis clip. ADA protein in Binding buffer (at pH 7.5, with 20mM Tris, 0.5M NaCl, and 30mM Imidazole) was transferred from 15mL falcon tube into the dialysis tube. The air bubbles were excluded from dialysis tube. Top of dialysis tube was folded twice and sealed with another dialysis clip.
    • The same process was performed for the protein purified on 11/06/2012. About 10cm of dialysis tubing was used for ADA elution fraction 1 and 3, and about 5cm of dialysis tubing was used for ADA elution fraction 2.
    • One Slide-A-Lyzer Buoys Dialysis Cassette was attached with groove of buoy on edge of one of the dialysis clips to ensure the ADA solution in dialysis tube float in water.
    • All three dialysis tubes were placed in water in dialysis beaker with stir bar.
    • Dialysis beaker was covered with aluminum foil to prevent loss of water in the process, and the beaker was placed on a stir plate in -40°C for 12 days.
  • The Au/ADA solutions were made to ensure the ratio of fibers formed. The ratios of Au/ADA solutions were as follows:
  60 - 70 - 80 - 90 - 100 - 110 - 120 - 130 - 140 - 150
  • The preparation for Au/ADA solutions were as follows:
    • ADA used in this set of samples had a concentration of 58.37uM and was made from 10/03/2012.
    • The HAuCl4 stock solutions was made on 09/05/2012 and has a concentration of 10.50mM.
    • The water used was distilled water at 16 mega ohms.
    • Below table listed the amount of ADA, HAuCl4, and water added for samples for all ratios:



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