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