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WHOLE CELL PROTEIN EXTRACTS

 

WHOLE CELL PROTEIN EXTRACTS

This if for both cytoplasmic and nuclear proteins-if you want only nuclear follow nuclear extract protocol.

  1. Lyse cells (1X107) in 1 ml. of the following buffer:
  2. 20 mM Tris pH 7.5

    100 mM NaCl

    0.5% NP-40

    0.5 mM EDTA

    0.5 mM PMSF

    0.5% protease inhibitor cocktail (Sigma)

    pipet cells up and down extensively and then sit on ice for 10 minutes.

  3. Spin down extracts full speed in microfuge for 10 minutes at 40C.
  4. Transfer supernatant carefully into a new tube.
  5. Perform Bio-Rad protein assay.
  6. ALTERNATIVELY-FOR VERY INSOLUBLE (i.e. cytoskeletal proteins etc.)---

  7. Lyse 107-108 cells in 250-500 ml of cell extraction buffer by pipetting up and down and then sonicating briefly to shear DNA (microtip for about 10 sec.).
  8. Determine protein concentration using Pierce BCA Protein Assay.
  9. Load 20 mgs (small gel) or 100 mgs (large gel) of total lysate onto SDS-PAGE gel-Western blot if necessary.

 

Cell extraction buffer

20 mM Tris pH8.0

100 mM NaCl

1 mM EDTA

0.1% SDS

1 mM PMSF

1X protease inhibitor cocktail