Biology Laboratory Techniques/Cell Fractionation

Cell Fractionation

Purpose

Cell Fractionation is a standard procedure used to separate subcellular organelles based on how cells differentially separate in a centrifuge. Organelles separate in a centrifuge based on density, shape, and size. The nucleus is typically the first organelle to become pelleted in a centrifuge.

The central piece of equipment in cell fractionation is a centrifuge. A centrifuge is a piece of equipment that spins rapidly and thus adds a centripetal force on the object that is spinning. All particles in the centrifuge will feel the same force being applied, but those that are more dense will move much faster through the liquid and sediment with less force than particles that have lower densities.

The centrifugal (or centripetal) acceleration is usually expressed as multiples of the acceleration of gravity. For example, if the centrifuge is exerting the same force that gravity would exert, then it is expressed as 1 G. If it's exerting a force 10 times stronger than gravity, then it is expressed as 10 G. The relative centripetal acceleration (commonly called the Relative Centrifugal Force)can be calculated using the formula RCF= (r X RPM² X (2π)² * (100/36))/(1000² X g) Where:

• r = the radius in centimeters of the sample from the center of the centrifuge
• RPM = Revolutions Per Minute
• g = gravitational constant (9.8 m/s²)

This equation is usually reduced to RCF = 11.19 X r X (RPM/1000)²

If your centrifuge will not obtain the specified RCF, then you may be able to compensate by centrifuging for a longer time. For example, if a procedure calls for centrifuging with an RCF of 10,000G for 60 seconds, but your centrifuge can only obtain 9,000 G, you may be able to compensate for this by centrifuging for 67 seconds. The equation to determine the compensation time required is: t_r = (t_s X RCF_s)/RCF_max Where:

• t_r = the time required in your centrifuge
• t_s = the time specified in the instructions
• RCF_s = The RCF specified in the instructions
• RCF_max = The maximum RCF your centrifuge can obtain

Differential Sedimentation:

Separation of cellular organelles is possible because particles tend to sediment at different rates. There are several factors that determine the sedimentation rate.

• Density: All other things equal, the more dense a particle is, the faster it will sediment. However if a particle is the same density as the liquid in the sample, then it will not sediment at all. If, however, the particle is less dense than the liquid medium (as is the case with oils and lipids when placed in water), it will move to the top of the medium.
• Size: All other things equal, the larger a particle is, the faster it will sediment. If two rocks of different size, but the same density are dropped off of a boat, the larger rock will tend to sink faster. This is because there is more mass (assuming density is equal) on which gravity can act. The larger mass can more easily overcome the friction of the water and sink faster. This does not mean that gravity acts differently on different masses. It is simply another way of describing the action of the medium that it is passing through. A single feather will fall slower than a bag of feathers. However, in a vacuum, they would fall at the same speed.
• Viscosity: The viscosity (or thickness) of the liquid medium can slow the rate of sedimentation. If two particles are relatively close in size and density and only have small differences, it may be possible to separate them from each other by increasing the viscosity of the liquid medium.

This procedure allows isolation of the cell nucleus, mitochondria, and cell membrane.

Required materials

Reagents

Fractionation Buffer

• 250 mM Sucrose (~4.28g in 50 mL)
• 20 mM HEPES (pH 7.4)
• 10 mM KCl (0.0373 g in 50 mL)
• 1.5 mM MgCl₂
• 1 mM EDTA
• 1mM DTT
• Protease Inhibitor (1X concentration)

• NOTE: Add the DTT and Protease Inhibitor At time of use only!

Lab Equipment

• (1) 10-cm Plate
• (3) 1.5-mL Eppendorf tubes (exact number of tubes depends on which fraction is desired)
• (1) 25 Ga needle
• (1) 1-mL syringe
• (1) Container of Ice (incubation)
• (1) Set of micropipettes
• (1) Sonicator
• (1) Centrifuge capable of:

   --- 720 G (for nuclear fraction) 
   --- 10,000 G (for mitochondrial fraction)
   --- 100,000 G (for cell membrane fraction)