In commenting on the use of Percoll gradients for the isolation of nuclei, Eric
".. Although I have not isolated nuclei from roots I have purified
them from arabidopsis leaves and from tomato fruit. Percoll is indeed
helpful, however a large percentage of the nuclei will penetrate even 80%
percoll. To overcome this problem I have run the crude nuclei extracts through a
percoll step gradient; from top to bottom 40%:60%:80%:2.2M sucrose. Most of the
nuclei pellet at the sucrose:80% percoll interface,however, some may be found
pelleted at the bottom of the tube, as told by DAPI staining. In both places I
found starch crystals. ..."
I am surprised that nuclei will penetrate 80% Percoll; perhaps nuclei of higher
plants are more dense than those of other organisms. Another possibility is that
2.2 M sucrose produces a very high osmotic potential, such that water is
withdrawn from the nuclei, and their density inevitably increases.
There are two additional strategies, which involve "suPercoll" and Ludox AM.
Ellen Reardon and I found that the density of Percoll can be substantially
increased by pressure dialysis, but "suPercoll," as we called it, is no longer
stable to heat sterilization.
Ludox AM is another formulation of silica sols, more reactive and less stable to
pH, but significantly more dense. I enclose a table of gradient materials for
comparison. We have described the use of Ludox AM in the isolation of
chromoplasts in Meth. Plant Cell Biol. vol. 50B, pp. 189-207 (Galbraith,
Bourque, & Bohnert, eds., Academic Press,1996).
A final point: because "Percoll" is a semi-eponym, it should be capitalized. It
was invented by Hakan Pertoft at the University of Uppsala, and Pharmacia
distributed the original preparations under the name "Pertoft's Colloid," which
was later condensed to "Percoll."
C. A. Price
Piscataway, NJ 08855-0759, USA
price at mbcl.rutgers.edu
<<<<<< Attached TEXT file named "Gradient.Mats" follows >>>>>>
Physical properties of some gradient materials.
Tab-separated columns are:
Concentration of stock solution
Density of stock solution
CsCl 60.0% w/w 1.7900 20 - -
D2O 100% 1.105 20
Ficoll 46.5%w/w 1.1629 4 1020
Glycerol 100% 1.2609 20 1490 20
Ludox AM 30.1%w/w 1.206 16 25
Metrizamide 85% w/v 1.466 5 246 5
Sorbitol 60%w/w 1.2584 4 102.9 4
Sucrose 65%w/w 1.32600 4 56.5 20
Percoll 100% 1.13 10 20