Tag / protein extraction

by Gabriele Feigelman

How to Prepare Lung Lysates from Mice Lung Tissue

  1. Label Eppendorf tubes.
  2. Prepare the Lysis Buffer by mixing 270 µL with 30 µL of Protease inhibitors (Sigma Cat. No S8820, 10x) for one lung tissue measuring approximately 2×2 mm. The ratio should be 1:10. Calculate the required volumes based on the number of specimens:
  • N of specimens x 300 µL = Final Volume
  • N of specimens x 30 µL = Required volume of protease inhibitors
  • Final Volume PBS = Final volume – Required volume of protease inhibitors
  1. Sonicate specimens on ice using short pulses not exceeding 5 seconds.
  2. Incubate specimens on ice for 30 minutes to cool down.
  3. Centrifuge at 12,000 RPM for 15 minutes.
  4. Collect fresh supernatant in a separate Eppendorf tube.
  5. Determine the protein concentration using the Bradford Assay (see Protocol for the Bradford Assay.

    PRO TIP: Maintain optimal cooling conditions during lung tissue preparation, sonication, centrifugation, and supernatant collection