Protein Extraction

Ben Carter, 7/11/2016

Required Materials
BioSpec Mini-Beadbeater-96

20mm diameter zirconia beads (BioSpec Cat# 11079124zx, can be washed and re-used)

2mL screw-cap tubes (pop-cap tubes will not work with the bead beater)

Bio-Rad RC DC Protein Assay kit (Cat# 5000122)

cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Sigma Cat# 4693159001)

Procedure
Note: Killer Buffer needs to be prepared fresh every time!''

1. Label a 2mL screw-cap vial and fill it about ½ full with the zirconia beads. Fill the tube about ¾ full with Killer Buffer. Ensure that the beads are mixed with the buffer so that no air bubbles remain.

2. Harvest tissue and fill the top half of the tube fully. Add Killer Buffer to submerge the tissue completely and make a convex solvent bead at the mouth of the tube. Air left inside the tube will cause the sample to aerosolize and should be avoided. I often stuff as much tissue into the top of the tube as possible, so over-loading the sample is not likely a concern.

3. Screw the cap onto the tube. Place all samples in the bead beater tube rack and perform the bead beating as per manufacturer’s instructions for three minutes.

4. Remove tubes from bead beater and transfer as much solution as possible to labeled 1.5mL microcentrifuge tubes. The beads can be washed and re-used if desired.

5. Centrifuge the solutions at maximum speed for 5min to pellet insoluble debris. Transfer the supernatant to a new labeled 1.5mL tube and discard the pellet.

6. Divide the protein samples into ~100uL aliquots and place in the -80°C freezer. This prevents repeated freeze/thaw cycles that can degrade proteins stored in Killer Buffer.

7. If desired, measure concentration of the samples using the RC DC Bradford assay kit as per the manufacturer's instructions. Note that it must be an RC DC kit due to the presence of both SDS and DTT in the Killer Buffer.

Killer Buffer

 * 800mg Sucrose (8%)
 * 400mg SDS (4%)
 * 1.2g Urea (2M)
 * 77mg DTT (50mM)
 * Dilute to 10mL with Milli-Q water.
 * Add one cOmplete Mini protease inhibitor tablet and rock until dissolved.