- Aluminum Foil
- Liquid Nitrogen
- Dry Ice
- 5 mL Culture Tubes
- Phosphate Buffered Saline
- Protease Inhibitor Cocktail (Sigma Cat.no. P-8340)
- Quant-iT™ Protein Quantitation Kit (Cat. no. Q33210)
1. Remove liver aseptically.
2. Flash-freeze liver in foil packets using liquid nitrogen. Store at –80°C.
3. Crush liver sample on dry ice and store in a prechilled 5 ml culture tube.
4. Add about 100 μg tissue (enough to almost cover round portion of a 5 ml culture tube) to a new chilled tube.
5. Add 1 ml PBS with 10 μl protease inhibitors (Sigma Cat. no. P-8340). Chill this solution using wet ice.
6. Homogenize at low speed for ~20 seconds. Make sure to keep cool and keep samples on wet ice.
7. Transfer samples into 1.7 ml microcentrifuge tubes.
8. Centrifuge at 14,000 x g at 4°C for 15 minutes.
9. Remove and aliquot supernatant. We recommend making several 50 μl aliquots.
10. Before running ELISA, dilute protein 1:100 and run a Bradford total protein assay or Quant-iT™ Protein Quantitation Kit assay (Cat. no. Q33210).
11. Dilute the samples to equal concentrations and then run ELISA as described under ELISA Protocol.
For Research Use Only. Not for use in diagnostic procedures.