This document discusses techniques for sub-cellular fractionation, specifically differential velocity centrifugation and density gradient centrifugation. Differential velocity centrifugation separates cellular components based on size, shape, and density by centrifuging at progressively higher speeds. Density gradient centrifugation separates components based on density by layering a cell extract on top of a buffer solution with a gradient of increasing density, allowing components to separate out as they migrate to the position matching their own density. The document provides details on procedures and materials needed to perform sub-cellular fractionation using these techniques.

1. Sub-cellular fractionation

2. Importance of separating sub-cellular fractionation
 To study about a particular subcellular component, we need the sample in
large amount.
 This technique enable us to separate pure samples of subcellular components
in reasonably active states.
 Successful approach for large number of proteomic studies is cellular
fractionation.
 Study of metabolic activity of the components – Mitochondria &
Peroxisomes.
 A well-established technique for separation of organelles is solely based on
two different types of centrifugation, differential velocity and density
gradient centrifugation making use of differences in sedimentation
coefficients and densities.

3. Density gradient centrifugation
 Separates sub-cellular components based on their density
 A non-ionic molecule like sucrose, glycerol is used to vary the density of the
buffer solution.
 The concentration of the molecule is varied and therefore the density of the
solution and the concentration is greatest at the bottom of the centrifuge tube
and decreased slowly at the top.
 A step gradient can also be prepared by using a series of solutions of different
concentrations – layered on the top one another with highest concentration at
the bottom.
 The cell extract is layered at the top of the gradient and the tube is subjected to
centrifugation at high speed for several hours.
 During this time, the different sub-cellular components move down the tube
until they reach the position in the density gradient that corresponds to the
density of the component.

5. Differential velocity centrifugation
 Separates cellular components based on size/shape and density.
 Larger more dense components will pack at the bottom of a centrifuge tube
faster and at lower speeds than smaller less dense ones.
 Initially, the total cell extract is centrifuged at a low speed for a short time
to remove unbroken cells.
 Later, the speed and time of centrifugation are progressively increased
removing some components. (Packing them at the bottom of the tube as
precipitate) and leaving others in the supernatant at each step.
 The final supernatant after the step-wise removal of nuclei, mitochondria,
vacuole, ribosomes which is called soluble fractions containing soluble
proteins and other small molecule components of the cytosol such as
tRNAs.

7. Materials required
 Homogenizing buffer:
 0.25 M sucrose
 1 mM EDTA
 10 mM Tris-HCl (pH 7.4)

8. Homogenize the cells in the buffer prepared as mentioned above in homogenizer.
Procedure
Centrifuge at 1000 g for 10 min
Pellet - nuclei
Supernatant (Centrifuge at 15000 g for 15 min)
Pellet – Mitochondria, Lysosomes
& peroxisomes
Supernatant (Centrifuge at 100000 g for 60 min)
Pellet – Vesicles & Microsomes
Supernatant Cytosol