Arabidopsis thaliana nuclei purification protocol

I obtained this protocol from Yuelin Zhang's group at the National Institute of Biological Sciences (NIBS, China). It has been published in the MOS7 paper (get the full reference from the Publications page):

 

Cheng  Y.*, Germain  H.*, Wiermer  M.*, Bi D.*, Xu F., García A., Wirthmueller  L., Després  C., Parker  J., Zhang  Y., and Li X. 2009. Nuclear Pore Complex Component MOS7/Nup88 Is Required for Innate Immunity and Nuclear Accumulation of Defense Regulators in Arabidopsis. The Plant Cell (In press).

 

Note that all steps must be carried on ice

 

Buffers :

 

Lysis Buffer (LB):

 

20 mM            Tris-HCl pH 7.4

25%                 Glycerol

20 mM            KCl

2 mM              EDTA

2,5 mM           MgCl2

250 mM          Sucrose

 

Add 1 mM PMSF just before use.

 

Nuclear resuspension buffer with Triton (wash buffer):

 

20 mM            Tris-HCl pH 7.4

25%                 Glycerol

2,5 mM           MgCl2

0.2%               Triton X-100

 

Procedure:

 

  1. Grind 1 g of healthy A.thaliana leaves to a fine powder in liquid nitrogen.
  2. Homogenized in 2 volume of Lysis buffer with PMSF.
  3. Filter successively the homogenate on a 100, 70 and 30 micrometer nylon mesh
  4. Centrifuge at 1500Xg 10 minutes to pellet nuclei. Supernatant can be kept, its cytosolic fraction.
  5. Wash pellet with 1 volume of wash buffer at least three times or until no green color can be seen.
  6. Resuspend final pellet directly in 100 microliter 1X Laemli buffer and boil for 5 minutes (if Western is the next application).