TISSUE DISRUPTION PROTOCOL

D. BOTTARO, UOB/NCI

March 18, 2009

1. Collect tissue samples for cryostorage and protein extraction in screw-top micro tubes (Mikro-schraubröhre, 1.5 ml PP; Sarstedt, Cat # 72.692.005); thaw frozen tissue samples on ice.

2. Add lysis buffer to the sample tube with the goal of achieving a 1.0 mg/ml or greater protein concentration (estimating buffer volume relative to tissue mass may require pilot experiments; we use a ratio of 3:1, buffer vol:tissue vol), then glass beads (0.5 mm glass beads, Biospec Products, Inc. Cat # 11079105) at a ratio of 1:6, bead vol:total volume.

3. Put the tube(s) in the bead beater (Mini Bead Beater 8, Biospec Products, Inc.) and set the speed to maximum for 30 sec, then remove the tube(s) and place on ice 40 sec. Repeat this 3 times.

4. Centrifuge sample(s) @ +4C, 14,000 rpm (Eppendorf Centrifuge 5417R) for 15 min.

5. Transfer supernatant(s) to new tube and determine protein concentration.

TISSUE DISRUPTION PROTOCOL

PROTOCOL REVISION APRIL 2010

Materials Needed/Preparation

  1. Lysis Buffer prepared on the same day tissues are to be processed
  2. Labeled o-ring tubes for tissue lysate storage
  3. 0.5 mm Glass Beads (Soda Lime, Cat No 11079105, BioSpec Products)
  4. Fast Cool centrifuge to 4˚ C (ensure no tubes were left inside)

Lysing Tissue & Harvesting Protein

  1. Allow samples to thaw on ice (It is important to keep samples at 4˚ C while they are being processed)
  2. Once samples are thawed:

Ø  For large tissue samples flash frozen, proceed to step 3

Ø  For small tissue samples frozen in Lysis Buffer, proceed to step 4

  1. For large tumor samples, cut a sufficient piece of tumor (~50 μl)

Ø  Place the excess tumor in the original vial and refreeze at -80˚ C immediately

Ø  Place the tumor sample in a clean o-ring tube

Ø  Add ~ two times the volume of sample of Lysis Buffer (~100 μl)

  1. Carefully add ~ 10 – 15 glass beads to the tube
  2. Load tubes into the bead beater and secure the lid tightly
  3. Ensuring the bead beater speed is set to zero, turn the power switch to the on, rather than timed, position
  4. Slowly bring the speed to the maximum setting
  5. Allow the samples to shake for 20 seconds at maximum speed
  6. Bring the speed back to zero and quickly place the tubes on ice to let them cool
  7. Repeat the shaking process two more times for a total shaking time of 1 minute per sample.
  8. Spin shaken samples at ~10,000 to 14,000 rpm for ~10-15 minutes at 4˚ C
  9. Transfer supernatant to new, labeled o-ring tubes for storage at -80˚ C
  10. Determine Total Protein concentration using BCA Protein Assay (tissue lysates generally require at least a 10-fold dilution to be within the range of the BCA assay)
  11. Measure diluted lysate in the appropriate assay or store protein lysate at -80˚ C