A Practical Guide to Cancer Systems Biology

28 A Practical Guide to Cancer Systems Biology

Equipment

• Magnetic stir plate and stir bar


• Mortar and pestle


• 1.5 mL centrifuge tubes


• Benchtop refrigerated centrifuge


• Homogenizer (LABSONIC M ultrasonic homogenizer; Sartorius AG)


• Vortexer


• Protein LoBind Tubes, 2 mL (Eppendorf)


• Heating block


• pH-Fix test strips (pH-Fix 0-14, MACHEREY-NAGEL, cat. no. 92110)


• Tweezers


• Centrifugal evaporator (CVE-2000; Eyela)


• Parafilm


• Water bath


• Syringe filters (0. 2 μm)


• ZipTip Pipette Tips (Millipore)

Reagent setup

• 1M Tris-HCl (30mL)

Dissolve 3.6342 g of Tris-base in 10 mL of Milli-Q water and stir on a
magnetic stir plate. Adjust the pH to 6.8 with 6M HCl and add Milli-Q
water to bring the final volume to 30 mL. Store it at room temperature.

• 100 mM Tris-HCl (30 mL)
  Mix 3 mL of 1 M Tris-HCl with 27 mL of Milli-Q water. Store it at room
  temperature.


• 1 ×Lysis buffer (20 mL)

10% Sodium dodecyl sulfate (SDS) 2 mL

100 mM Tris HCl 10 mL

Glycerol 2 mL

Milli-Q water 6 mL

Aliquot 1 mL and store at− 30 ◦C. Add protease inhibitor just before use.

• 200 mM Triethylammonium bicarbonate buffer (TCEP)
  (200μL)
  Dissolve 11.45 mg of TCEP in 200μL of Milli-Q water. Freshly prepare
  this reagent and keep it at 4◦Cbeforetheexperiment.