Rat Liver Preparation

互联网2013-11-13

1055

实验试剂

1. Aluminum Foil

2. Liquid Nitrogen

3. Dry Ice

4. Phosphate Buffered Saline

5. Protease Inhibitor Cocktail (Sigma Cat.no. P-8340)

6. Quant-iT™ Protein Quantitation Kit (Cat. no. Q33210)

实验步骤

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.

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