Sample Preparation for SDS-PAGE
NOTE: For some cell lines (like CMT3-COS) resuspension in water and sample buffer is sufficient. For other cell lines (like 293 and 293T) the DNase is necessary to decrease the viscosity.
For a typical 100 cm plate
- Spin down the washed cell plate in an eppendorf tube.
- Resuspend the pellet in 100ul of 5mMCaCl2 and 10mM MgCl2 in water. Be sure to get an even suspension.
- Add 100 ul 2X sample buffer. The sample will become viscous as the cells and nuclei lyse. Pipet up and down to make the solution as uniform as possible.
- Add 5 ul (5 units) RQ DNase (Promega). Pipet up and down some more to mix.
- Let sit about 15 minutes at RT.
- Boil 5 minutes.
- Centrifuge briefly to remove undissolved material. From some cell lines there will be no pellet. From other cell lines (like 293T) the pellet will be very small.
- Load the supernatant on the gel.
As a general rule you should never load more than 1/5 of a plate (40ul). This will vary according to the cell line.