CELL FRACTIONATION
Created
by
Daniel Leuthardt
12/13/93
This protocol is intended for use with 4x 162 cm2 flasks that have reached 80 % confluence. When following this procedure, it is important to keep everything at 4 0c at all times.
Cell Preparation
- Wash each flask with 10 ml of PBS 3x
- Add 10 ml of PBS to each flask
- Gently scrape bottom of flasks 2x with large scraper to remove cells
- Put suspension into a 50 ml centrifuge tube
- Centrifuge at about 1000 rpm, 4 0c, 5 min.
- Vacuum off supernatant and re suspend pellet in cold Hypertonic solution (4 0c)
- Homogenize with 10 stokes of homogenizer
- Take 2.1 ml of homogenate and add 0.7 ml of 1 M sucrose to make it 0.25 M with respects to sucrose. Put the rest of the homogenate in a tube, label it Whole Cell Fraction, and freeze it.
- Centrifuge the homogenate/sucrose mixture at 11,000 rpm, 4 0c, 10 min.
Supernatant
- Spin the supernatant at 35,000 rpm, 4 0c, 1 hour.
- Transfer supernatant to a different tube, label, and freeze.
- Re suspend the Precipitate in 100 m of PBS, label Membrane Fraction, freeze.
Precipitate
- Re suspend precipitate in 250 m of Isotonic solution.
- Layer sample over 250 m of 1.7 M Sucrose in a tube.
- Centrifuge at 14,000 rpm for 1 min.
- Label supernatant Boundary Fraction, freeze.
- Re suspend precipitate in 100 m of Isotonic solution, label Nuclei Fraction, freeze.
Solutions
Hypertonic solution
25 mM MES ( pH 6.2 )
25 mM Sucrose
1.5 mM MgCl2
0.1 mM CaCl2
2 mM PMSF
2 mg/ml Leupeptin
_______
5 ml
Isotonic solution
0.75 ml Hypertonic solution
0.25 ml 1 M Sucrose
______
1 ml