Chapter 3: Cell Fractionation - Endnotes

  1. Bean sprouts or alfalfa sprouts from a local supermarket may be substituted. They are inexpensive, non-poisonous and contain immature chloroplasts which separate well upon sucrose gradient centrifugation.

  2. For optimal performance, the sucrose concentration should be determined by use of a refractometer. Pure sucrose, manufactured for use in molecular biology should be used, or it may be necessary to treat the sucrose for contaminating enzymes.

  3. Modified from Terrance G. Cooper. The Tools of Biochemistry. John Wiley & Sons, New York, 1977. pp 347-352.

  4. If only one laboratory section is used, two of each gradient should be prepared. The extra are used to balance the tubes in the centrifuge. It is not good practice to balance an ultracentrifuge with water or differing gradients since the density distribution will not be balanced, even if the total weight is.

  5. This rotor has served the author well over many years, but is no longer available. The SW27 rotor has a k = 337 @ 25,000 RPM. Refer to Appendix F for conversion directions for other rotors.

  6. Continuous flow spectrophotometry may be substituted, if available.

  7. Data from Techniques of Preparative, Zonal, and Continuous Flow Ultracentrifugation by Owen Mitch Griffith, Ph.D. Applications Research Department, Spinco Division, Beckman Instruments, Inc. 1979, page 9.

  8. Ibid, page 4.

  9. These beads are a quick means of visually checking the density separation during formation of the Percoll gradient. They are available from Pharmacia, but are expensive. The use of the beads may be eliminated, with the corresponding elimination of Step 1a.

  10. As reported in Pertoft, H., et al. "The Use of Density Gradients of Percoll^R for the Separation of Biological Particles." in Separation of Cells and Subcellular Particles (H. Peeters, Ed.) Pergamon Press, Oxford, 1979.

  11. Sepratech Corporation, Oklahoma City, OK 73127.

  12. Sepracell-MN tubes were devised for human whole blood, but will work well on rabbit or other animal blood. The blood needs to be less than 12 hours old for best results, and no older than 36 hours.

  13. If a fixed angle rotor is unavailable, use a swinging bucket, but increase the centrifugation time to 20 minutes.

  14. Miller, R.G. and R.A. Phillips. "Separation of Cells by Velocity Sedimentation" J. Cell Physiol., 73:191-202. 1969.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 --