Preparation of Tissue Samples:

1. Thaw sample on Ice if frozen or put new sample on ice.

2. Weight sample in microtube.

3. Add appropriate Lysis Buffer to sample.

4. Grind or homogenize sample on ice.

5. Vortex samples on medium speed (10-20min).

6. Sonicate sample: Power 3; 20 sec., three times with 10 sec. between sonications.

7. Spin at 6000RPM, for 3 min. Save supernatant, store pellet at -20°C.

8. Clean up sample with 2D Clean Up kit (Amersham) via procedure from manufacturer.

9. Add appropriate Lysis buffer to sample: (50-100)µL per tube.

10.Vortex on medium speed for 30 min.

11. Combine split tubes.

12. Centrifuge samples at 14000RPM for 20 min.

13. Save supernatant.

14. Determine protein concentration with 2D-Quant Kit (Amersham).