MadSci Network: Biochemistry
Query:

Re: What is the necessary g force for isolating mitochondria in a centrifuge?

Date: Sun Sep 9 19:35:46 2007
Posted By: Neil Saunders, Computational biologist
Area of science: Biochemistry
ID: 1184192073.Bc
Message:

Dear Catherine,

Thanks for your question - amateur science in your spare time is a very admirable pursuit!

Let's look at the basics of centrifugation. Left to themselves, particles above a certain mass and density will eventually settle to the bottom of a tube. The keyword here is "eventually". The time that particles take to settle is given by Stoke's Law:

vt = 2R2s-ρ)a/(9μ)
This states that the velocity of the particle depends on its radius, the difference in density between the particle and the medium in which it is suspended, the viscosity of the medium and the acceleration applied to the particle. Mitochondria are relatively large particles compared with cells, but if we try some numbers for a mitochondrion in this equation (radius ~ 1-2 x 10-6 m, density ~ 1100 kg m-3), water density 1000 kg m-3 and water viscosity 8.9 x 10-4 kg m-1 s-1, we can see that a mitochondrion will take many days to sink to the bottom of a tube under normal gravity (9.8 m s-2). So a centrifuge works by increasing the acceleration felt by the particle many-fold.

There are two basic approaches to cell fractionation. The first is called differential centrifugation and relies primarily on the variation in size (radius) of the cellular components. Using this method, the sample is spun repeatedly, reserving the supernatant and increasing the g-force each time. Unbroken cells, debris and nuclei will pellet first at about 1000 g. Mitochondria, chloroplasts and lysosomes will pellet at around 10 000 g. In this type of centrifugation, increasing the time can compensate for lower centrifugal acceleration. For instance, 20 minutes at 10 000 g will have approximately the same effect as 10 minutes at 20 000 g.

The type of centrifugation that you refer to, isopycnic centrifugation, is a sub-class of a method called density gradient centrifugation. There are two ways to do this. The first is called rate zonal centrifugation. To do this, you place layers of a solution (commonly sucrose) in the centrifuge tube, starting with high density at the bottom and ending with low density at the top. Centrifugation through the step-wise density gradient rapidly seperates the particles based on their radius and mass. Some rotors are more suitable than others for this purpose and you have to be careful not to centrifuge for too long, or everything pellets at the bottom.

Isopycnic centrifugation is subtly different. You suspend the material to be centrifuged in a concentrated solution - commonly cesium chloride or polymeric substances called Percoll or Ficoll - and centrifuge at very high g-force, such as 100 000 g. Under these conditions, the solution forms a linear density gradient along the tube. Particles sink until they reach the point where their density equals the surrounding density in the solution, at which point further centrifugation has no further effect. This method is most commonly used to separate macromolecules (such as DNA and RNA) or ribosomes. The high g-force is required to generate and maintain the linear density gradient.

So to answer your question: 20 000 g is not sufficient for isopycnic centrifugation - but technically, what you want to perform is rate zonal centrifugation. Your centrifuge is more than capable of pelleting mitochondria and I think if you experiment with linear sucrose gradients (and depending on your starting material), you should able to obtain a reasonably-pure fraction of mitochondria. Here are some references to help you:

Best of luck with the experiments,
Neil


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