Retour vers UQTR
Chaire de recherche du Canada en immunité végétale
Génomique fonctionnelle des phytopathosystèmes

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

Note that all steps must be carried on ice

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).