Cell lysis (urea)

Lysis Buffer

  • 8 M Urea
  • 40 mM NaCl
  • 50 mM tris
  • 2 mM MgCl2
  • 50 mM NaF
  • 50 mM b-glyceradelhyde phosphate
  • 1 mM sodium orthovanadate
  • 10 mM sodium pyrophosphate
  • 1 (or part of one depending on volume) mini EDTA-free protease inhibitor (Roche Diagnostics)
  • 1 (or part of one depending on volume) phosSTOP phosphatase inhibitor (Roche Diagnostics)

Procedure

Lysis

  1. Add this to pelleted cells in a ration of ~3:1 (buffer to pellet). Pippette up and down to resuspend pellet.* We often add a little benzonase too to digest the DNA and decrease the viscosity of the solution.
  2. Sonicate (on ice) 30 seconds on, 60 seconds off (repeat 3 times total)
  3. Spin max speed on benchtop centrifuge to pellet debris
  4. Keep supernatant (determine protein concentration)

Digestion (trypsin)

  1. Add LysC in a ratio of 1:100 (enzyme:protein) (~2 hours at 37 °C)
  2. Dilute with 50 mM tris pH 8.0 until urea concentration is 1.5 M
  3. Add trypsin in a ratio 1:50 (enzyme:protein) (overnight at 37 °C)