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Equipment and reagents

Lysis buffer

  • 50 mM Tris-HCl pH 8.0
  • 1% DMSO
  • 50-200 mM NaCl*
  • 1 mM EDTA
  • 1 mM PMSF
  • 1 µg/ml leupeptin
  • 1 µg/ml pepstatin A

*The NaCl concentration used in the lysis buffer depends fully on the application. In case of affinity chromatography on a Ni-column the NaCl concentration is usually 200 mM but when the first purification step is ion exchange chromatography no salt should be added.

Stock solutions

  • 100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol
  • 1 mg/ml leupeptin in water
  • 1 mg/ml pepstatin A in methanol

Equipment

  • Vortex mixer

Preparation of glass beads

  • Glass beads (diameter 500 µm)
1. Soak the glass beads for 16 h in concentrated HCl.
2. Rinse thoroughly in distilled water.
3. Bake them for 16 h at above 150°C.
4. Chill the glass beads at 4°C or on ice before use.

Procedure

This procedure is suitable for small samples (up to 15 ml). For larger samples, specialized apparatus have been developed, such as Biospec Products Bead Beater, which allows cell suspensions up to 200 ml to be subjected to agitation at once.

1. Prepare the glass beads by washing them in concentrated HCl, followed by extensive rinsing (check that then pH is neutral) and drying. The dried beads should be chilled before use.
2. Resuspend the cells in an equal amount of chilled lysis buffer and place the suspension in a sturdy tube (preferably not glass).
  • Add the PMSF (10 µl PMSF (100 mM) per ml of celsuspension) at this point.
3. Add 1 - 3 g of chilled glass beads per gram of cell wet weight.
4. Vortex 3 - 5 times for 1 minute, each time keeping the cells on ice for 1 minute between vortexings. use the highest setting of the vortex mixer.
5. Remove the glass beads.
6. Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).