Whole Cell Extract of Tetrahymena cells

''This protocol outlines the steps for lysing Tetrahymena cells under denaturing conditions. For Lysis under native conditions, proceed with the same except your buffer of choice for lysis will be different (it will not contain chaotropic agents like Urea or Guanidine). ''

Materials

 * Tetrahymena cultures (100 mL to 1 L are commonly used; protocol is the same with any volume used)
 * Starvation Buffer (10 mM Tris-HCl, pH 7.5)
 * Urea Lysis Buffer (8 M Urea; 100 mM Na2HPO4-NaH2PO4 pH 8.0; 10 mM Tris-HCl pH 8.0; 5 mM Imidazole)
 * N-ethylmalemide (NEM)
 * Protease Inhibitors (commonly used ones are antipain, chymostatin, E-64 and leupeptin)
 * Spin Bottles (appropriate sized for volume to be lysed)
 * Sonicator (Broyles Lab)
 * High speed microcentrifuge (volumes up to 2 mL) and ultracentrifuge (volumes >2 mL)

[OPTIONAL] Induction of Cells with Cadmium

 * 1) Induce cells with 0.1 ug/uL cadmium for 2 hours and place in shaking incubator at 30 °C.

Lysing Cells

 * 1) Spin down 100 mL of cells corresponding to OD540 0.3 at 1,100×g for 3 minutes.  If you are lysing 500 mL or 1 L of cells, simply scale up the amount of Urea Lysis Buffer used in the lysis step #4. For volumes >2 mL you will use an ultracentrifuge instead of a microcentrifuge. 
 * 2) Wash twice with Starvation Buffer, decant the supernatant quickly and then resuspend in Starvation Buffer again and p lace cells at 30 °C in a shaking incubator and allow to starve for 5-10 minutes.
 * 3) Spin cells again at 1,100×g, decant the supernatant and then immediately lyse by adding 1 mL of Urea Lysis Buffer (with NEM and protease inhibitors). Place on ice immediately.
 * 4) Sonicate samples by giving 30 pulses at (setting 7 and output 50%). Wait 30 seconds and give 30 more pulses.   Wear noise-cancelling head-set to prevent ultrasonic waves that may cause hearing loss. Ensure the sonicator tip is cleaned when lysing different samples. Do NOT touch energized Sonicator tip against any surface as that may break the tip. Check to see if the cells are lysed by placing 10 uL on a slide or plate and using a microscope; intact cells not should be observed.  
 * 5) Pipette lysates into microcentrifuge tubes and spin at highest setting at 4 C for 1 hour. Pipette out 100  uL of clarified lysate into a microcentrifuge tube. When working with larger volumes, use an ultracentrifuge to prepare lysates. typical run times are 1-2 hours. Consider transferring the lysates into a conical tube and centrifuge again at high speed in a swinging rotor centrifuge as this has been shown to clear lysates if there is still cell debris present.
 * 6) Prepare clarified lysates for SDS-PAGE run by adding 4×SDS Loading Buffer and DTT (final conc. 1 mM).