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:
- The filters were placed in 1M NaOH and sonicated for one hour.
- The filters were then placed in dH2O and sonicated for another hour.
- The filters were placed in ethanol and sonicated for a half hour.
- Finally, the filters were sonicated again in dH2O for a half hour.