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Tissue Preparation Protocols for Membranes and Lysates

Simple protocols for preparing membrane fractions and tissue lysates.

Tissue preparation is incredibly important and dramatically affects the success of subsequent experiments. Here, we describe simple protocols to prepare membrane fractions and tissue lysates.

Enriched Membrane Fraction Preparation

  1. Remove the tissue of interest from the animal and flash freeze it in liquid nitrogen. Store the tissue at -80°C until further use.
  2. Place the frozen tissue in 5 volumes of ice-cold Lysis Buffer A (4 mM HEPES (pH 7), 320 mM sucrose, 5 mM EDTA (pH 8), and Complete EDTA-free protease inhibitor cocktail (Roche)).
  3. Homogenize the tissue with a polytron homogenizer.
  4. Centrifuge the homogenate for 10 minutes at 2,000 x g at 4°C. Discard the large debris.
  5. Transfer the supernatant to a clean tube and resuspend the pellet in 2 volumes of Lysis Buffer A and rehomogenize.
  6. Centrifuge the homogenate for 10 minutes at 2,000 x g at 4°C. Combine the supernatant with that from step 5.
  7. Centrifuge the supernatants (from steps 5 and 6) for 1 hour at 100,000 x g at 4°C.
  8. Discard the supernatant. Resuspend the pellet (that contains tissue membranes) in 2 volumes of Lysis Buffer A and briefly homogenize with a polytron homogenizer.
  9. Measure the protein concentration using the Bradford method. Adjust the protein concentration to 4 mg/ml with Lysis Buffer A.
  10. Store protein samples at -80°C until further use.

Lysate Preparation

  1. Remove the tissue/organ of interest from the animal and flash freeze it in liquid nitrogen. Store the tissue/organ at -80°C until further use.
  2. Place the frozen tissue in 5 volumes of ice-cold Lysis Buffer B (50 mM Tris (pH 7.4), 5 mM EDTA (pH 8), 1% Triton X-100, and Complete EDTA-free protease inhibitor cocktail (Roche)).
  3. Homogenize the tissue with a polytron homogenizer.
  4. Rotate the sample for 30 minutes at 4ºC.
  5. Centrifuge the homogenate for 1 hour at 100,000 x g at 4°C.
  6. Transfer the supernatant to a clean tube and measure the protein concentration using the Bradford method. Adjust the protein concentration to 4 mg/ml with Lysis Buffer B. Store the lysate at -80°C until further use.