Lysis and immobilization

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  1. Suspend one cell pellet ( ) in 5 mL of storage buffer ( ) with 1 mg/mL #lysozyme and one cell pellet without lysozyme.
  2. Place the suspended pellets in the -80°C for 15 mins.
  3. Thaw the mixture with hands until all the ice has melted.
  4. Repeat steps 2 and 3, two more times.
  5. Centrifuge mixture to remove cell debris.
    • Separated cell lysate into eppendorfs (1 mL per) and centrifuged for 30 mins at 13 krpm
  6. Mix cell lysate with 63 μm SiO2 at a ratio of 250 μL per 5 mg for 30 mins
    • Have sample container lie on its side to maximize surface area exposure.
    • Used 1 mL of cell lysate with 20 mg of silica
    • Had samples lie flat on a shaker
  7. Spindown mixture and remove the supernatant.
  8. Washed the silica-immobilised protein with 1 mL of storage buffer per eppendorf, twice.
  9. Suspend silica-immobilised protein at a ratio of 25 μL storage buffer to 5 mg silica.
  10. Stored all the immobilise protein in one eppendorf at 4°C

Results: All the silica used was very pink. Immobilisation appears to have been effective. Final volume of approx. 800 μL.

Lysozyme with freeze-thaw appeared to be more effective than freeze-thaw without lysozyme.