8. Isolation of peroxisomes
Van Veldhoven, P. P., Baumgart, E. and Mannaerts, G. P.
(1996) Anal. Biochem., 237, 17-23
• Light mitochondrial fraction layered on a
20-40% (w/v) iodixanol gradient
• Centrifuged at 100,000g for 1h
• Gradient unloaded dense end first
10. Fraction number
% Distribution Density (g/ml)
1 3 5 7 9 11 13 15
0
5
10
15
20
25
30
1
1.05
1.1
1.15
1.2
Catalase
Succinate
dehydrogenase
ß-Galactosidase
Light mitochondrial fraction in 30% iodixanol, layered under a 20-
27% iodixanol gradient – 70, 000 g for 1.5 h
11. Fraction Number
%Distribution
Density(g/ml)
1 3 5 7 9 11 13 15 17
0
5
10
15
20
25
30
1.1
1.12
1.14
1.16
1.18
1.2
1.22
1.24
Density Succ deHase Catalase ß-Gal'ase
Purification of peroxisomes (mouse liver) in a self-
generated iodixanol gradient: LMF in 25% (w/v)
iodixanol: 10 ml fixed-angle (20°) 180,000 g(av) for 3 h.
12. Fraction Number
%Distribution
Density(g/ml)
1 3 5 7 9 11 13 15 17 19
0
20
40
60
80
1.06
1.08
1.1
1.12
1.14
1.16
1.18
1.2Density Gal trans Catalase
ß-Gal'ase Succ deHase
Fractionation of mouse liver LMF in a self-generated
iodixanol gradient: LMF in 15% (w/v) iodixanol: 10 ml
fixed-angle (20°) 180,000 g(av) for 3 h.
13. Fraction Number
%Distribution
Density(g/ml)
1 3 5 7 9 11 13 15 17
0
10
20
30
40
50
60
1.06
1.08
1.1
1.12
1.14
1.16
1.18
1.2
Density Succ deHase Catalase
ß-Gal'ase Gal trans
Fractionation of mouse liver LMF in a self-generated
iodixanol gradient: LMF in 17.5% (w/v) iodixanol: 10 ml
fixed-angle (20°) 180,000 g(av) for 3 h.
14. Fraction Number
%Distribution
Density(g/ml)
1 3 5 7 9 11 13 15 17 19
0
10
20
30
40
50
60
1.08
1.12
1.16
1.2
Succ-deHase Catalase ß-Gal'ase
Density Gal Trans
Fractionation of mouse liver LMF in a self-generated
iodixanol gradient: LMF in 20.0% (w/v) iodixanol: 10 ml
fixed-angle (20°) 180,000 g(av) for 3 h.
15. Dissection of lipid-rich domains inDissection of lipid-rich domains in
iodixanol gradientsiodixanol gradients
Adapted from Lindwasser, OW and Resh MD (2001) J. Virol., 75, 7913-7924
10%
40%
50%
30%
20%
Caveolin
Cholesterol
GM1
Na+
/K+
-ATPase
16. Exosome purification strategy (I)
Clarify culture medium
300-800 g 10-15 min
800-10,000 g 10-30
min
10-20,000 g 10-30 min
and/or
filter (0.1-0.45μm)
Clarify culture medium AND serum
100,000 g; 1-16 h
and/or 0.22μm filter
Culture
Concentration of exosomes
by pelleting
100-150,000 g 1-2 h
12,000 g; 70,000 g, 100,000 g
Iodixanol density gradient
Today, homogenates of cultured cells are often only centrifuged once at a low speed to remove the nuclei prior to loading on to a gradient. Although this speeds up the separation process and ensures minimal losses of any potentially important small organelles and/or vesicles, the vast array of particles remaining in the supernatant will severely test the resolving power of any density gradient. In some respects therefore the “old-fashioned” approach of carrying out a full differential centrifugation schedule prior to any density gradient method has its own merits.
Density of solutions only depends on the solute concentration and its atomic composition. Atomic composition of iodixanol and Nycodenz virtually identical; similarly for sucrose and polysucrose. Compare this with the effect of concentration on solution osmolality (next slide).
Osmotic pressure is dependent solely on the NUMBER of molecules per ml of solution. Density gradients that are iso-osmotic with plasma and with the cytosol are very desirable. The osmolality of plasma is approx 290 mOsm (dashed horizontal line). Above approx 8.5% sucrose (1.032 g/ml) solutions become grossly hyperosmotic (green line). All osmotically sensitive particles lose water in sucrose gradients and approach a limiting density. Osmolality of polysucrose solutions is much lower; the molecular mass a polysucrose molecule is at least 1000x greater than that of a sucrose molecule so in solutions of the same % w/v concentration there will be 1000x fewer molecules of polysucrose, compared to sucrose – hence the much lower osmolality. The profile of the polysucrose osmolality is not linear because at concentrations above 20%, water molecules become sequestered by the polysucrose molecules, so increasing their effective concentration. The osmolality profile of Nycodenz (red line) is more or less linear and a solution of approx 29% (w/v) Nycodenz in water is iso-osmotic. Because iodixanol has approx. double the molecular mass of Nycodenz, the osmolality is about half that of Nycodenz (dark blue line). If however you measure the omsotic pressure of OptiPrep (60% iodixanol in water) the OP is lower than expected. This is because, in aqueous solutions, iodixanol associate non-covalently to form oligomers – hence the effective number of particles is reduced. However when OptiPrep is diluted with any isoosmotic buffer, the iodixanol the oligomers disperse and the solutions behave as shown by the blue line. Thus when OptiPrep (60% iodixanol) is diluted with a buffered saline solution (or any other solution which is itself iso-osmotic) all the iodixanol solutions produced will be iso-osmotic. This makes handling OptiPrep for the separation of any particle very easy.
Because of the isoosmotic nature of iodixanol gradients all osmotically sensitive organelles have a lower density in iodixanol than in sucrose. The density of mammalian nuclei in sucrose is not known as these organelles will pellet through solutions of the highest concentration possible. The limiting membrane of peroxisomes is permeable to small molecules and their densities in sucrose and iodixanol overlap
It is not possible to band nuclei during their purification in a sucrose gradient. They will pellet very slowly through dense highly viscous sucrose solutions that have a sky-high viscosity. Hence the need for long centrifugation times and high speeds.
Note the much lower g-force and centrifugation time needed for a three-layer iodixanol gradient. The nuclei band at the lower interface and the entire gradient is isoosmotic.
Flotation of mitochondria (from a heavy or light mitochondrial fraction adjusted to approx 1.2 g/ml) through a discontinuous gradient. Lysosomes band at the top interface, mitochondria at the middle and peroxisomes at the bottom interface. If it is required to isolate the study the lysosomes as well then a small layer of homogenization medium should be added to the top – this prevents particles banding at an air/liquid interface. Beause mitochondria are particularly sensitive to hydrostatic pressure, which is greatest at the bottom of the tube, the crude fraction is often loaded in the middle layer (far right)
Only peroxisomes are sufficiently dense to sediment into the gradient, the rest of the organlelles band across the original sample/gradient interface.
A simple shallow gradient can resolve all the major particles in a liver light mitochondrial fraction. The sharp drop in density at the top of the gradient occurs because of a layer of homogenisation medium is added to the top.
First of a series of four graphics devoted to a self-forming iodixanol gradient. The more shallow middle section effectively separates the peroxisomes. These four gradients also show that some small volume fixed-angle rotors (with a relatively low angle) can be effective in the formation of self-generating gradients
The next three graphics show the effect of small increases in the starting concentration of iodixanol on the distribution of Mouse liver Golgi, lysosomes, mitochondria and peroxisomes in the same rotor
See comments to slide 24
See comments to slide 24
A very rarely quoted separation of detergent solubilized particles was published by Resh’s group at the Sloane-Kettering in New York. It is a multiple flotation layer gradient which suggests that rafts exist as a heterogeneous group of domains that appear to be characterized different ratios of the raft markers (caveolin, cholesterol and the GM1 glycolipid
