Rat Liver Preparation

Materials

  • 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)

Protocol

  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.

BioSource   C-070276 1107      1-Jan-2007