User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/29

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Triple Mutant Purification

  • The previously expressed and extracted Asc Hb M8S/M33S/M103S protein was syringe filtered.
  • 1L of 25mM Tris, 50mM Nacl, pH 8 buffer was prepared (3.03g Tris + 2.92g NaCl, used HCl to pH, diluted to 1L with dH2O). This buffer was vacuum filtered in room 207.
  • When the FPLC was attempted to be used with our buffer, the pressure increased too much and the purification needed to be stopped (before the protein was even loaded).
  • The filter on the FPLC was changed to a new one but the pressure still increased too much so the purification was abandoned for the day.
  • The 25mM Tris, 50mM NaCl buffer was re-filtered using a vacuum but in Dr. Harting's lab instead.
  • Previously used filters for the FPLC were cleaned in the following way:
    1. The filters were placed in 1M NaOH and sonicated for one hour.
    2. The filters were then placed in dH2O and sonicated for another hour.
    3. The filters were placed in ethanol and sonicated for a half hour.
    4. Finally, the filters were sonicated again in dH2O for a half hour.