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

  1. Spin down the washed cell plate in an eppendorf tube.
  2. Resuspend the pellet in 100ul of 5mMCaCl2 and 10mM MgCl2 in water. Be sure to get an even suspension.
  3. 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.
  4. Add 5 ul (5 units) RQ DNase (Promega). Pipet up and down some more to mix.
  5. Let sit about 15 minutes at RT.
  6. Boil 5 minutes.
  7. 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.
  8. 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.