Chloroplasts were successfully separated by centrifugation
in a sucrose density gradient into particle
fractions using a zonal rotor.1 Since then, various
aspects of the procedure have been used with different
swinging-bucket rotors. This technical article
describes the large-volume F10-6x500y rotor with
conical bottle and adapter assembly (Figure 1) that
was used in the Vortex 21K high-speed centrifuge
(Figure 2) for all centrifugal sample preparations
(FIBERLite Centrifuge Inc., Santa Clara, CA).
The conical bottles in the adapters (250 mL) were
used in place of swinging-bucket rotors for density
gradient separations and to collect sediment in the
small cone area of the conical bottles (Figure 3).
Standard round-bottom bottles were not used with
the fixed-angle rotors.
Figure 1 - F10-6x500y rotor with conical bottle and
Figure 2 - Vortex 21K centrifuge.
Figure 3 - Conical bottle and adapter assembly.
Methods and results
About 400 g of market spinach leaves were deveined
and blended for 15 sec in 500 mL of 0.4 M sucrose,
0.05 M tricine buffer at pH 7.4. The homogenate
was filtered through cheesecloth, and the filtrate was centrifuged for 10 min at 4000 × g (5000 rpm). The
pellet was resuspended in 250 mL of 0.3 M KCl, 0.05
M tricine, at pH 7.7, then pressed through a French
pressure cell (SLM Instruments Inc., Urbana, IL)
three times at 12,500 psi. The resulting homogenate
supernatant was centrifuged for 10 min at 4000 ×
g (5000 rpm) to remove the larger cell fragments.
The pellet was again diluted with 250 mL of 5% w/w
sucrose to make 250 mL homogenate sample containing
about 6.0 mg chloroplast per milliliter in the
5% w/w sucrose.
Each conical bottle was loaded with a stepwise
sucrose gradient of 70 mL per step made up in 0.15
M KCl and 0.05 M Tris buffer, pH 7.7. The sucrose
concentrations were layered in the centrifuge conical
bottles using a Pasteur pipet that reached to the
bottom of the bottle. The lightest step of the gradient
of 10% concentration was placed in the bottle
first. The second concentration, 30%, was layered
below the 10%, and finally the 50% concentration
was layered below the 30%. Care was taken not
to disturb the interfaces between the layers by
holding the tip of the pipet to the wall of the
bottle when removing it or introducing it into
the conical bottle.2
Immediately after the concentrations were layered
into each bottle, 40 mL of the homogenate
sample was layered in the top of each gradient to
prevent premature sample or gradient diffusion.
The bottles were then placed in the adapters and
installed in the rotor cavity. A slow acceleration/deceleration profile for the centrifuge was chosen to
prevent sample gradient mixing during acceleration
and or deceleration. The maximum speed of (10,000
rpm) 17,000 × g at 4.0 °C with a run time of 2.5 hr
was selected for the chloroplast separation.
At the end of the run, three fractions were visually
observed, and they were removed from each bottle
by pipet. Spectrophotometric measurements of the
fractions were made at 680 nm. Fraction 1 at the top
of the bottle was removed first. This fraction represented
nongreen light scattering material. The visible
green fraction 2 in the center zone of the bottle
contained chlorophyll and was removed, and fraction
3 in the cone of the bottle that contained the
chloroplasts was removed. Each fraction was diluted
at a ratio of 1:2 with buffer and centrifuged at 4000
× g for 10 min to collect the sediment. Table 1 shows
the distribution of chloroplast in the three fractions.
The results presented in Table 1 show that good
separation of the fractions was accomplished, and a
substantial amount of each fraction was collected in
a single operation with the F10-6x500y rotor. The
conical bottles for all centrifugal sample preparations
including the density gradient separation of
the sample separation made it easy to determine the
relative amounts of chloroplast in each fraction. The
simplicity of use of the Vortex 21K high-speed centrifuge
was also noted.
The large-volume fixed-angle rotor with conical
bottles can be used successfully in place of a large-volume
swinging-bucket or zonal rotor to obtain
good separations by density gradient centrifugation.
- Brown, J.S.; Griffith, O.M. Year Book; Carnegie Institution:
Stanford, CA, 1971, 69; 705.
- Griffith, O.M. Practical Techniques for Centrifugal Separations. FIBERLite Centrifuge, Inc.: Santa Clara CA, 2006; Vol. 1; p 40.
Dr. Griffith is Director of Research, FIBERLite
Inc., 422 Aldo Ave., Santa Clara, CA 95954,
U.S.A.; tel.: 408-988-1103; fax: 408-988-1196; e-mail: Mgriffith@piramoon.com.