gradients are often used for separations and purifucations of cells, viruses
and subcellular particles. To keep these in good condition, the gradient medium
should have the following characteristics:
- Cover a sufficient density range for isopycnic
banding of all biological particles of interest.
physiological ionic strenght and pH
be iso-osmotic throughout the gradient
to penetrate cell membranes
able to form self-generated gradients by centrifugation at moderate g-forces
with biological materials
- Be easy to remove from purified materials
- Have no quenching effects on radio activity
- Have no adverse effect on assay procedures
of the density gradient media that fulfils these criteria is Percoll. This
medium is based on colloidal silica and it is coated with polyvinylpyrrolidone
(PVP). The percoll particles have diameter of 15-30nm and can form a gradient
in the range 1,0-1,3 g/ml. Percoll is delivered sterile and each bottle has a
known mean density and osmolality.
Generating a percoll gradient
decide with which density, the so called starting density, you want to start.We always generate a Percoll gradient in the
presence of 0.15M NaCl. The following formula gives the quantity of Percoll
needed when working volume and starting density are known.
volume of Percoll (from the bottle) in ml
volume of final working solution in ml
desired density of final solution in g/ml
density of Percoll (from the bottle, see bottle label) in g/ml
density of 1.5 M NaCl = 1.058 g/ml
calculated volume of Percoll, add 1/10 of the final working volume of 1.5 M
NaCl (i.e 10 ml for 100 ml working volume), fill up to the desired working
solution with sterile water and mix this well (Note: final concentration of NaCl=0.15M).
- Divide the working solutions over centrifuge tubes.
Keep enough room in the tubes for adding the cell suspension at a later time
(i.e. 8 ml in 10 ml tubes).
the calculation of the shape of the generated gradient, we add marker beads to
the tubes. These are 9 kinds of freeze-dried cross-linked dextran beads having
an accurately determined density in Percoll with 0.15M NaCl. Add 1 ml sterile
water to each vial and allow the beads to swell overnight. Make a mix of the
swollen beads: add 50 Ál of each vial to 0.5 ml water in a Eppendorf tube. Mix
well and add 40 Ál to the tubes. Place the tubes in the SS34 angle rotor of
the Sorvall centrifuge. Centrifuged the tubes for 45' minutes at a rotor speed
of 15000 rpm to preform a gradient.
Figure 2.9: Shape of the Percoll gradient after 5' (A), 15' (B),
30' (C) and 45' (D) centrifugation. The dashed line marks the starting density,
i.e. the "shape" of the gradient at the beginning.
shows how the gradient is generated during centrifugation. The gradient forms an
S-curve and this is a very important
feature of the gradient.
flat part of the curve equals roughly the density of the cells, we obtain the
best separation of cells on the basis of small density differences. The
preformed gradient is very stable and can be stored for several days in the
- Load the preformed gradient with cells. Concentrate
10 ml steady culture of OD450=0.2 to 0.5 ml and pipette it carefully on top of the gradient
and close the tubes. Place the tubes in the swingout rotor and centrifuge for
15 minutes at 9000 rpm.<
centrifugation a milky cell band is visible in the gradient. If the starting
density equals the density of the cells, the band is broad and in the middle of
the gradient. If the density of the cells is higher or lower then the starting
density we find a small band in the steeper part of the gradient.
- After these two centrifugation steps, we can
calculate the shape of the gradient and the density range of the cells.
Measure the distance from the different marker beads to the bottom of the tube
and plot the values as in figure 2.9. Do the same with the bottom and top of
the cell band. Draw at these values a vertical line in the graphic and the
intersection with the curve of the gradient gives the density range of the
- With help of a fraction collector is it easy to
fractionate the cell band. Small unbudded yeast cells have a low density and we
can find them in the first fractions. These fractions we can we use for a
synchronization, but the disadvantage is that the cell concentration is low and
the fraction is not so pure as we can get with centrifugal elutriation, i.e.
the fraction contains more dividing and parent cells. This is the reason why we
use Percoll gradients only for density measurements.
- With help of a Percoll gradient it is easy to
seperate different cells with a different density. We have to choose a starting
density lying between the densities of these cells.
1.5 M NaCl and Percoll
|Sharpe, P.T., 1988, Methods of cell separation, Laboratory techniques
in biochemistry and molecular biology. Elsevier science publishing company.|
|Pharmacia: info-book about Percoll.|