Separation of Spinach Chloroplasts Using a Large-Volume, Fixed-Angle Rotor

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 adapter assembly.

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.

Conclusion

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.

References

  1. Brown, J.S.; Griffith, O.M. Year Book; Carnegie Institution:
    Stanford, CA, 1971, 69; 705.
  2. 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 Centrifuge Inc., 422 Aldo Ave., Santa Clara, CA 95954, U.S.A.; tel.: 408-988-1103; fax: 408-988-1196; e-mail: Mgriffith@piramoon.com.

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