PREPARING WHOLE CELL LYSATES

1. Pellet cells at 500 x g for 3 min. (Make sure you pre-weigh your tubes so you can determine a cell weight)
2. Remove supernatant and wash 3-4 times in wash buffer. Remove all supernatant and re-weigh cells to obtain a wet weight.
3. For whole cell lysates, resuspend cells in one of the recommended 2D sample buffers. It is easy to try 2 or 3 buffers and run a 1D gel to see which method gives you the most proteins.
   • a. Add a minimum of 500 µL lysis buffer for each 100 µL cells
   • b. OR add enough lysis buffer to yield approximately 50 mg/ml (So if have 15 mg of cells, then add 300 µL lysis buffer). Vortex or briefly sonicate the sample and let it remain at room temp for 30 minutes. DO NOT heat samples containing urea above 30° C. Protease inhibitors and/or endonucleases can also be added, especially if you do not intend to further clean the sample. Add DTT (or you can use TBP) fresh each time. Let the sample sit at room temperature for 20-30 minutes to completely solubilize the proteins.
4. Spin 10,000 x g for 10 minutes at 4C. Alternatively, you can ultracentrifuge the sample for 30 minutes at 60,000 x g to separate the soluble and insoluble fractions.
5. Place the supernatant (soluble material) in a fresh tube and discard the pellet (insoluble material). Use immediately or store at -70°C.