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General Cell and Tissue Lysis

*Choose 1st step depending on what you are lysing and proceed to 2nd step*

Step 1: [Cell Lysis]
  1. Set Plates on Ice
  2. Wash plates with cold PBS two times
  3. Add fresh lysis buffer onto plates (on a 100 mm plate: 0.5mL for fibroblasts and 1 mL for adipocytes
  4. Scrape cells from plates
Step 1: [Tissue Lysis]
  1. Completely thaw out tissue from the -80. Chop tissues into small pieces.
  2. Mix enough lysis buffer to cover the tissue. Homogenize tissue with dounce homogenized 20 times.

Step 2:
  1. Rotate lysate at 4 °C or 30 minutes
  2. Spin down cells by 14K rpm for 20 minutes
  3. Carefully remove fat cake from top using a needle and syringe, transfer supernatant into clean tubes and discard the pellet of unbroken cells and debris
  4. Measure Protein Concentration
  5. Mix 20-60 μg of lysate with 1/4V of 4x LDS sample buffer
  6. Boil samples for 5 minutes