1. Wash w/ 1mM CaSO4
2. Wash w/extraction buffer (0.1M K-Pi, ph 7.5, 5mM EDTA, 1mM DTT)
3. Frozen in –80C
4. Grind tissues w/ eppendorf pestles
5. Centrifuge at 14000g for 10min
6. Aliquot supernatant
7. Determine protein concentrations
8. In 0.1ml supernatant, add 0.4ml of ddH2O, 1ml of 1% (W/v) sulfanilamide in 2N HCL and 1ml of 0.1% NED in ddH2O
9. Develop for 30min
10. Determine OD at 540
11. Against standard curve