General Cell and Tissue Lysis
*Choose 1st step depending on what you are lysing and proceed to 2nd step*
Step 1: [Cell Lysis]
Set Plates on Ice
Wash plates with cold PBS two times
Add fresh lysis buffer onto plates (on a 100 mm plate: 0.5mL for fibroblasts and 1 mL for adipocytes
Scrape cells from plates
Step 1: [Tissue Lysis]
Completely thaw out tissue from the -80. Chop tissues into small pieces.
Mix enough lysis buffer to cover the tissue. Homogenize tissue with dounce homogenized 20 times.
Rotate lysate at 4 °C or 30 minutes
Spin down cells by 14K rpm for 20 minutes
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
Measure Protein Concentration
Mix 20-60 μg of lysate with 1/4V of 4x LDS sample buffer
Boil samples for 5 minutes