SDS Lysis
Whole-cell protein extraction of tissue culture cells can be obtained by using a simple buffer solution containing SDS (sodium dodecyl sulfate) and Tris base. The SDS acts as an anionic detergent that will cause cells to lyse and expell their nuclear and cytoplasmic extracts while the Tris base will keep the pH of the buffer stable. This method of protein isolation will not give a "pure" protein extract. If purity is important, more elaborate protocols must be used.
SDS Lysis buffer:
Basic protocol
1. Isolate tissue culture cells
- for adherent cells:
- aspirate medium
- rinse cells with 20 mL PBS/ 10 cm plate
- treat with 2 mL trypsin-EDTA to detatch the cells from the plate
- add two volumes of complete medium with serum to the trypsin-EDTA treated cells, pipette up and down to break up clumps and transfer to a 15 mL centrifuge tube
- pellet cells in a clinical centrifuge (500 g for 5 minutes)
- aspirate the supernatant
- resuspend cells in complete medium containing serum (to inactivate the trypsin)
- pellet cells again and aspirate supernatant
- for suspension cells:
- transfer cells to a 15 mL centrifuge tube, pellet cells in a clinical centrifuge (500 g for 5 minutes)
- aspirate the supernatant
2. Add 2mL of boiling lysis buffer per 15 mL centrifuge tube, swirling the solution over the cell pellet to enure rapid denaturation of cellular protiens.
3. The lysate sample will be viscous due to cellular DNA.
4. Transfer the lysate to a 50 mL centrifuge tube.
5. Briefly heat the lysate (heating block or microwave) with tube uncapped.
This step should be monitered carefully, as there is a potential for sample loss due to spills.6. One of these methods can be used to shear the cellular DNA:
- sonicate lysate sample for 10-30 sec
- pass lysate sample 10X through a 25-gauge needle
- homogenize lysate sample with a polytron for 15-30 sec
7. Remove a 15 µL aliquot and add 1/5th volume (3µL)
6x SDS sample buffer, mix thoroughly and place in a boiling water bath for 2 minutes or heat block.
8. Store at -20C