Exercise 3.1 - Sucrose Fractionation of Castor Bean
Figure 3.4 Homemade gradient forming device
- Castor beans 1
- Gradient former Sucrose solutions in 0.01 M EDTA, pH 7.5
33%, 44%, 50%, 57%, 60% (w/v) 2
- Grinding Medium
- 0.4 M sucrose
- 0.165 M Tris-HCl buffer, pH 7.5
- 0.01 M KCl
- 0.01 M MgCl
- 0.01 M EDTA adjusted to pH 7.5
- 0.01 M dithiothreitol
- Chopping board and knife or razor blade
- Mortar and pestle
- Refrigerated centrifuge (ultracentrifuge preferred) with
swinging bucket rotor
- Spectrophotometer and tubes
- Hemacytometer and phase contrast microscope
- Germinate castor beans by soaking overnight, followed by
germination in moist vermiculite, or paper towels, at
Castor beans are poisonous and may be fatal if taken
internally. Wear gloves while handling.
- After approximately 5 days, remove the embryos and
cotyledons and discard. Wash the endosperm in distilled
water and chill on ice.
- Prepare two centrifuge tubes for sucrose gradients; one
for a linear 33-60% gradient and the other for a stepped
The volume of each solution will depend on the centrifuge
rotor to be used and its corresponding centrifuge tube
capacity. The following are directions for 35 ml centrifuge
Carefully layer, in order, the following solutions into a
- 3 ml of 60% sucrose
- 6 ml of 57% sucrose
- 9 ml of 50% sucrose
- 9 ml of 44% sucrose
- 3 ml of 33% sucrose
Add 5 ml of 60% sucrose to the bottom of a centrifuge tube
and form a linear 60 to 33% sucrose gradient on top of that.
Using a gradient former, as shown in Figure 3.4, place 12 ml
of 60% sucrose in the right chamber and 12 ml of 33% sucrose
in the left chamber.
- Combine 60 gm of endosperm tissue with 90 ml of grinding
medium and chop vigorously.
- Transfer the coarse material to a cold mortar and pestle
and continue to grind until a fine paste is formed.
- Filter the BREI (the paste from step 5)
through two layers of cheesecloth. Collect the fluid into a
beaker, transfer it to a centrifuge tube, and centrifuge the
filtrate for 10 minutes at 270 xg to remove unbroken cells
and large debris.
- Decant the supernatant into a clean, cold centrifuge tube
and recentrifuge for 30 minutes at 10,800 20xg. Resuspend
the pellet in 5 ml. of grinding medium and hold on ice for
- Carefully decant the supernatant and save for subsequent
analysis. Gently resuspend the pellet in 6 ml. of grinding
- Carefully layer 2 ml of the pellet onto a stepped sucrose
gradient and 2 ml of the pellet onto a linear gradient.
- Team up with another laboratory section and carefully
balance your corresponding tubes. That is, be sure that the
stepped gradients for both sections are exactly the same
weight. Add grinding media to balance, where appropriate.
Have the instructor check the balance before proceeding.
- Centrifuge the tubes at 4° C for 4 hours at
25,000 RPM in a Beckman SW27 rotor or equivalent. 5
- Upon completion of the centrifugation, fractionate the
samples into approximately 20-30 equal portions. It is
convenient to collect samples of 1.5 ml. This yields a
volume suitable for subsequent spectrophotometric analysis
without the use of small volume cuvettes.
This is accomplished most rapidly by gently inserting a long
18 gauge needle, with the tip ground off, into the
centrifuge tube so that it rests on the bottom of the tube.
Without moving the needle (so as not to stir the contents),
attach a 2.0 ml syringe and pull out the bottom 1.5 ml of
the sample. Remove the syringe and place the 1.5 ml fraction
into a test tube marked as #1. Re-attach the syringe without
disturbing the gradient, and repeat as often as need to
totally fractionate the gradient.
Alternatively, the gradient can be fractionated by
puncturing the bottom of the tubes with a needle, and
collecting the fractions in 1.5 ml portions by counting the
appropriate number of drops that drip from the punctured
tube. There are several commercially available fraction
devices designed for this purpose. If one is available,
follow the manufacturer's directions.
- Read the optical density (Absorbance) of each fraction
in a spectrophotometer at 540 nm. Plot a double graph, with
fraction number on the x axis (bottom to top) vs
OD on the y axis, and fraction number vs %
sucrose on the y axis. 6
- Carefully examine all fractions with a phase contrast
microscope. Identify and count (use a hemacytometer where
appropriate) all structures found in each fraction.
Graph of fraction number vs Absorbance
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org