Chapter 3: Cell Fractionation - Endnotes
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.
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
Modified from Terrance G. Cooper. The Tools of
Biochemistry. John Wiley & Sons, New York, 1977.
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
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.
Continuous flow spectrophotometry may be substituted, if available.
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.
Ibid, page 4.
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.
As reported in Pertoft, H., et al. "The Use of Density Gradients
for the Separation of Biological Particles." in Separation of
Cells and Subcellular Particles (H. Peeters, Ed.) Pergamon
Press, Oxford, 1979.
Sepratech Corporation, Oklahoma City, OK 73127.
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.
If a fixed angle rotor is unavailable, use a swinging bucket, but
increase the centrifugation time to 20 minutes.
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 -- email@example.com