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Glass Bead Method for Isolating Total Bacteria Proteins

  1. Weigh an empty tube and add 1.5ml exponential culture O.D. 600 Cells were pelleted and washed with  TDEP buffer.   The washed cells  are weighed and resuspended in 200 mL of ice-cold TDEP buffer. Add 1 mg prepared glass beads/mg wet cells to each tube. Each tube was vortexed for 30 sec.  with 30 sec. incubations on ice between vortexing , for a total of 4 times. The cell debris and DNA was removed by centrifugation for 10 min.  in microfuge.
  2. The supernatants were removed and stored at   -20°C.

  TDEP Buffer

10mM Tris. Cl, pH 7.4

0.5mM DTT

1.0mM EDTA

35mg/ml PMSF (0.2mM)

PRE-TREATMENT OF GLASS BEADS

  1. Add  1mL of concentrated HCl to 500mg beads in a microfuge tube.

  2. After 5 min. wash 5 times with dd H2O  (1mL each).

  3. Then wash glass beads twice with 1 mL of TDEP buffer.

  4. Remove as much liquid as possible, then add TDEP buffer to 1 mL total volume.