Hi, everybody out there!
Recently I am preparing nuclei from tomato leaves to get nuclear extracts.
As a start I followed a protocol from a friend. Leaf tissue is homogenized
in chilled homogenization buffer (10 mM MES pH 6.8, 10 mM NaCl, 0.25 M
sucrose, 5 mM EDTA, 0.2 mM PMSF, 0.15 mM spermine, 0.5 mM spermidine, 0.2%
Triton X-100) (1:3 w/v), filterd through 2 layers and 6 layers of
Miracloth respectively and then centrifuged at 2000x g for 10 min.
Precipitate is resuspended in 10 ml of homogenization buffer and
centrifuged again. Initially this washing step can effectively separate
nuclei from chloroplasts. After I ran out of the homogenization buffer I
had to prepare more following the recipe exactly the same as before.
Unfortunately the newly-made buffer could not allow the separation of
nuclei from chloroplasts, both nuclei and chloroplasts are precipitated. I
tried different sucrose concentrations from 0.25, 0.5, 0.75, and 1.0 M,
different pH from 6.0 to 7.0 and different salt concentrations from 10 to
50 mM. Even though I found pH is a factor of the most importance, no
unique pH can work as well as the homogenization buffer (pH 6.8) used
initially. It is so strange to me. I do not have any idea about what is
going on there.
I also tried to used Percoll step gradient (10%, 30% and 60%, or 30%, 60%
and 85%) to separate nuclei from chloroplasts. In all these trials,
chloroplast seems to be very heterogenous in density and distributed among
all three gradients although most chloroplasts floated on top of percoll.
I could not see any nuclear band. Could anybody out there have any
suggestions or working protocols to help me?