Rat Liver Preparation

• Aluminum Foil
• Liquid Nitrogen
• Dry Ice
• 5 mL Culture Tubes
• Phosphate Buffered Saline (Cat. No. 20012050)
• Protease Inhibitor Cocktail (Cat. No. 78429)
• BCA Protein Assay or 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. 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 BCA 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.

BioSource C-070276 1107 1-Jan-2007