Root nuclear extracts

[Mary Williams]

Dear Jose,

        I assume you mean Arabidopsis roots? I have made nuclear extracts
from Arabidopsis roots by growing the roots in liquid culture to get a
large mass of roots (root culture described in Williams and Sussex, Plant
J. 8, 65 - 76, 1995.) We used a protocol for small scale nuclear extracts
(Armstrong et al. Plant Cell 4, p 525 - 537).


We started with on average 10 flasks of roots and got:

starting weight                 mg protein
20 g                               0.6
12.5 g                             0.45
9.1 g                              0.45

        We were resuspending our nuclei in too much buffer (2 ml), so ended
up with  overly dilute nuclear extracts. We concentrated them by ammonium
persulphate precipitation and were then able to use them for gel shifts but
they were not concentrated enough for footprinting.
        I would advise starting with a HUGE amount of roots, which requires
patience....the Armstrong protocol works well but be aware that you want to
resuspend your nuclear pellet in the smallest volume possible. Also, the
nuclear pellet lies over a starch pellet - be careful to resuspend the
soft, easy -to -resuspend nuclei but leave behind the sticky, more opaque
starch pellet.

Good luck!

Mary Williams
Dept of Biology
Harvey Mudd College
Claremont, CA 91711