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RESEARCH DIVISION Laboratory Manual

 
 

Subcellular Fractionation of Proteins

  1. Plate 3 x 106 cells on a 10cm dish. Grow overnight.
  2. Harvest cells via scraping into ca. 500ul (per 10cm dish) CLB/5mM EDTA/PI. Allow cells to swell 5'. Bounce homogenize cells 50 times. Centrifuge 7500 rpm 5'. Pellet is nucleii plus debris. Supernatant is cytosol plus plasma membrane.
  3. Remove supernatant and spin in an SW41 rotor at 25000 rpm for 30'. Remove pure cytosol supernatant. Resuspend crude plasma membrane pellet in 40ul PBS.
  4. Resuspend pellet from step 1 in 1ml TSE/0.1% NP40/PI. Dounce homogenize 30 times and centrifuge 5000 rpm 5'. Resuspend pellet and wash twice. The pellet is very easily disturbed. Final pellet is pure nuclei. Resuspend in 50ul TSE/0.1% NP40/PI.

Buffers:

TSE:  (500ml)

10mM Tris pH 7.5 5ml 1M Tris pH 7.5
300mM sucrose 100ml 1.5M (102g sucrose/200ml)
1mM EDTA 1ml 500mM EDTA pH 8

CLB: (500ml)

10mM HEPES 5ml 1M (23.8g HEPES/100ml)
10mM NaCl 5ml 1M (5.84g NaCl/100ml)
1mM KH2PO4 500ul 1M (13.6g KH2PO4/100ml)
5mM NaHCO3 2.5ml 1M (8.4g NaHCO3/100ml)
1mM CaCl2 500ul 1M (14.7g CaCl2.2H2O/100ml)
0.5mM MgCl2 500ul 500mM (10.2g MgCl2.6H2O/100ml)

Protease inhibitors (PI):

1mM PMSF
10ug/ml aprotinin
10ug/ml leupeptin
1ug/ml pepstatin
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998