1. Fractionation of organelles and membrane vesicles using OptiPrep™
Competitive products are sucrose and Percoll®
Sucrose-based publications go back to 1948
The vast number of publications reporting the use of sucrose gradients for the fractionation of subcellular particles makes a change to OptiPrep often quite difficult, but there are some important advantages in using OptiPrep.

2. N P L M Golgi Exocytosis/secretion Caveolae Lipid rafts
Plasma membrane
Lipid rafts
Caveolae
Coated pits
Exocytosis/secretion
Endocytosis N M L P
Endoplasmic reticulum
Golgi
Trans-Golgi network
Endocytic
compartments
ERGIC
The huge number of subcellular membrane compartments underscores the complexity of the task to produce pure preparations of any one type of particle or the development of gradients that will allow identification of multiple particle types. This short training file will therefore deal principally with some general points about subcellular fractionation; describe a few of the commonly used organelle isolation methods and end with a few examples of analyses that demonstrate the resolving power of iodixanol gradients in studies of membrane trafficking and cell signalling. The next series of slides however (Slides 3-9) describe in general terms the operations that are carried out and some of the requirements and consequences of these operations.

3. Iodixanol gradient solution strategy (S01, S02)
Simple gradient solution preparation
When OptiPrep is diluted with the homogenization medium the solutions will be iso-osmotic
Step 1 is solution preparation. The use of OptiPrep makes gradient solution preparation as easy as possible – it may be simply diluted with a normal iso-osmotic tissue or cell homogenization medium and all the solutions produced will also be iso-osmotic.

4. Percoll® solution strategy
Because Percoll® has nearly zero osmolality the working solution must be made by diluting 9 vol of Percoll® with 1 vol of 2.5 M sucrose
2.5 M sucrose is close to saturation
2.5 M sucrose is impossible to handle accurately
Handling of Percoll is by contrast very difficult. Since Percoll has essentially no osmotic pressure, it is necessary first to produce a working solution by diluting 9 vol. with 1 vol. of 2.5 M sucrose. Firstly 2.5 M sucrose is difficult to prepare since it is close to saturation and it is so viscous that accurate volume dispensing is almost impossible.

5. Differential centrifugation of homogenate
Homogenize cells in 0.25 M sucrose,
1 mM EDTA, 10 mM Tris-HCl, pH 7.4
1000g/10 min
15,000g/15 min
supernatant
pellet
nuclei
Pellet (LMF)
mitochondria
lysosomes
peroxisomes
supernatant
100,000g/60 min
Step 2 is differential centrifugation of the homogenate. The slide shows a widely used protocol that precedes any gradient. Sometimes an additional step is inserted in which the first 1000 g is centrifuged at 3000 g for 10 min to produce the so-called heavy mitochondrial fraction (HMF). This supernatant is then centrifuged at 15,000 g to produce the light mitochondrial fraction (LMF).
In the analysis of ER, Golgi and plasma membrane, some workers will first prepare a microsome fraction as described in the slide, but some people will simply use the first 1000 g supernatant. The latter will contain microsomes plus all the mitochondria, lysosomes and peroxisomes.
pellet
vesicles (microsomes)
supernatant
cytosol

6. Carry out the density gradient centrifugation
Suspend the relevant pellet in a small volume of homogenization buffer (or dense solution)
Layer the suspension on top of (or below) the gradient
Centrifuge – usually in a swinging-bucket rotor
Collect the gradient in a series of fractions if required
Step 3 Following some form of differential centrifugation, the selected fraction is then applied to a gradient and in most cases, but particularly in membrane trafficking and cell signalling studies, the gradient will be collected in a series of equal volume fractions and analyzed

7. Analyze fractions
20-30 fractions ( ml each) need to be analyzed for some functional marker characteristic of each membrane and for total protein. Determine density of blank gradient
Measure marker enzyme spectrophotometrically OR do SDS-PAGE, electroblot and probe the blot with antibodies to marker proteins
Step 4 is analysis of the gradient fractions.

8. Density gradient medium should not interfere with analysis I
Percoll® is light scattering at all wavelengths so must be removed prior to spectrophotometric analysis
Percoll® must be removed prior to SDS-PAGE because it affects sample entry into gel
Removal of Percoll® particles leads to loss of organelles
Analysis of the fractions from a Percoll gradient pose important problems. The removal of Percoll means that each fraction is diluted and then centrifuged so that the Percoll particles form a pellet. The problem is that the g-force required to pellet the Percoll also causes the subcellular membranes to pellet – as a result significant losses of cellular material can be incurred.

9. Density gradient medium should not interfere with analysis II
Only for spectrophotometric analysis in the UV must iodixanol (or Nycodenz®) be removed
Removal of iodixanol (or Nycodenz®) does not lead to loss of organelles
Sucrose need not be removed prior to any analysis
In the great majority of cases none of these problems apply to gradients of Nycodenz or iodixanol or sucrose. All of these are true solutes and if necessary the membranes can be simply be pelleted from each gradient solution after dilution.

10. Four important examples of simple discontinuous iodixanol gradients
Purification of nuclei from a total homogenate
Purification of mitochondria from a crude mitochondrial fraction
Separation of cytosol and membrane vesicles
Isolation of lipid rafts
The methodology is so extensive and various that it is not feasible to cover more than a few specimen isolations in any detail – four of the most widely used fractionations are presented.

11. Purification of nuclei I (S08)
homogenate in
25% iodixanol
30% iodixanol
35% iodixanol
nuclei
10,000g
20 min
A prime example of the advantages of an iodixanol separation over a sucrose one is the isolation of nuclei. In this rapid and organelle-friendly method the whole tissue or cell homogenate is simply mixed with OptiPrep (3.5:2.5 v/v) and layered over two solutions of 30% and 35% iodixanol. Under very mild centrifugation conditions the nuclei band at the lower interface.

12. Purification of nuclei II
Advantages over sucrose
Density barriers of 60-65% sucrose - very difficult to prepare
Sucrose solutions very viscous, therefore need much higher g-forces ( ,000g and times (1-2 h)
Sucrose solutions are vastly hyperosmotic; only iodixanol allows nuclear isolation under iso-osmotic conditions
Iodixanol method: use whole homogenate, rather than nuclear pellet
Compare the easy and rapid iodixanol method with the traditional sucrose method, in which a 1000 g pellet from the homogenate is suspended in 60% sucrose and layered over 65% sucrose.

13. Density of particles in iodixanol allows superior resolution
Organelle
Sucrose
Iodixanol
Mitochondria
Lysosomes
Peroxisomes
Nuclei
>1.32
The isolation of many widely-investigated organelles poses problems for sucrose gradients. All the major organelles of the light mitochondrial fraction possess seriously overlapping densities in sucrose gradients. In part this is due to the much higher osmolality of sucrose gradients compared to those of iodixanol (or Nycodenz) – the organelles in sucrose gradients (in effect) approach a limiting density. It is for example necessary to lower the density of lysosomes artificially to resolve them in good yield from mitochondria.

14. Purification of mitochondria (S12)
Crude mitochondrial
pellet (1.204 g/ml)
1.079 g/ml
1.175 g/ml
Mitochondria
50,000g 4h
An example of a simple discontinuous iodixanol flotation gradient for the isolation of mitochondria

15. Isolation of peroxisomes I (S09)
Light mitochondrial fraction layered on a 20-40% (w/v) iodixanol gradient
Centrifuged at 100,000g for 1h
Gradient unloaded dense end first
Peroxisomes in particular benefit from the use of iodixanol (or Nycodenz) gradients – their purity is significantly higher compared to isolates from sucrose or Percoll gradients

16. Isolation of peroxisomes II
% Distribution
Density (g/ml) 1 3 5 7 9 11 13 15 17 19 10 20 30 40
1.05
1.1
1.15
1.2
1.25
1.3
Density
Glut deHase
Catalase
Acid Pase
G-6-Pase
63%
This simple linear iodixanol gradient, which can be executed in a fixed-angle rotor, resolves the peroxisomes from all the other major organelles in a light mitochondrial fraction. Only some residual endoplasmic reticulum (ER) can be detected. Yield and purity are both 90-95%. In Percoll the peroxisomes are much more seriously contaminated by ER.

17. LMF in self-generated gradient (S14)
Fraction Number
% Distribution
Density (g/ml) 1 3 5 7 9 11 13 15 17 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
Sometimes the aim of the gradient is not to isolate a particular organelle at the highest purity but to achieve a distinctive separation of all the organelles so that the location of some other function or component can be assessed. The ability of iodixanol to form self-generated gradients (see Slides of Training File 2) makes this a very easy task. In the example shown in this slide the light mitochondrial fraction is simply adjusted to a 17.5% iodixanol and centrifuged for 3 h in a suitable rotor.

18. Vesicle/cytosol separation (S36)
Crude vesicle
fraction
 1.16 g/ml
 1.05 g/ml
 1.14 g/ml
Vesicles
Cytosolic
proteins
,000g
1-3 h
The determination of whether a protein resides in the cytosol or is membrane-bound forms an important part of many subcellular membrane studies. A simple method is to add OptiPrep (or a dense Nycodenz solution) to a vesicle+cytosol sample to adjust it to approx 1.16 g/ml. Then to layer a 1.14 g/ml and 1.05 g/ml solutions on top. During the centrifugation the vesicles migrate to the top interface and the cytosolic proteins remain in, or start to sediment through, the sample zone. This is a much more efficient method than placing the sample on top of a density barrier.

19. N P L M Golgi Exocytosis/secretion Caveolae Trans-Golgi network
Endoplasmic reticulum
Plasma membrane
Caveolae
Coated pits
Exocytosis/secretion
Endocytosis N M L P
Golgi
Trans-Golgi network
Endocytic
compartments
ERGIC
Lipid rafts
The plasma membrane contains many specialized sub-domains and one of these, the lipid raft, is commonly separated in an iodixanol gradient

20. Isolation of detergent-insoluble membranes (S33)
Iodixanol conc.
20%
35%
40% HM
PNS
Lipid rafts
160,000g
4 h
The simple standard methodology is to isolate rafts as detergent-insoluble (detergent-resistant) membranes. Triton X100 (or some other non-ionic detergent) is included in all the solutions and a post-nuclear supernatant is adjusted to approx. 40% iodixanol. Two lower concentration iodixanol solutions and a small volume of homogenization medium (HM) are layered on top and during the centrifugation the rafts float to the top interface. Sometimes the 20% OR 35% iodixanol layer is omitted. The reproducibility of the iodixanol gradient is better than that of the corresponding sucrose gradient.

21. Dissection of lipid-rich domains in iodixanol gradients Adapted from Lindwasser, OW and Resh MD (2001) J. Virol., 75,
10%
40%
50%
30%
20%
Caveolin
Cholesterol
GM1
Na+/K+-ATPase
Iodixanol gradients also score significantly over sucrose gradients in their ability to resolve different types of lipid-rich raft domain. More sophisticated gradients can distinguish cholesterol-, caveolin- and GMI glycolipid-rich domains.

22. N P L M Golgi Exocytosis/secretion Caveolae Lipid rafts
Plasma membrane
Endoplasmic reticulum
Golgi
Trans-Golgi network
Lipid rafts
Caveolae
Coated pits
Exocytosis/secretion
Endocytosis N M L P
Endocytic
compartments
ERGIC
The majority of OptiPrep publications are concerned with the analysis of the complex pathways of membrane trafficking and cell signalling. This involves the construction of gradients that can achieve at least a partial resolution of many compartments – endoplasmic reticulum, endoplasmic reticulum Golgi intermediate compartment (ERGIC), Golgi, trans-Golgi network, early endosomes, late endosomes, plasma membrane, transport vesicles etc. It is not useful to present the this vast methodology. The remaining slides will merely present a few illustrations of the efficacy of the many types of gradient used.

23. High resolving power (S19)
Fraction number
% Maximum
Density (g/ml) 1 3 5 7 9 11 13 15 17 19 20 40 60 80
100
1.02
1.04
1.06
1.08
1.1
1.12 PM
Golgi ER
One of the first published papers to present the use of OptiPrep reports on the separation of the three major compartments, plasma membrane (PM), Golgi and endoplasmic reticulum (ER).
From Yang, M et al (1997) J. Biol. Chem., 272,
CHO cell PNS on 0-26% iodixanol gradient: 200,000g for 2h

24. High resolving power (S23)
Calnexin
ßCOP
Rab8
Fraction # 1 3 5 7 9
In this sedimentation velocity separation of microsomal vesicles, calnexin shows the location of the ER, ßCOP, the cis-medial Golgi and Rab8 the trans-Golgi network.
Mouse neuroblastoma cell; 3000g supernatant,
discontinuous gradient (2.5-30% iodixanol: 126,000g for 30 min.
From Petanceska, SS et al (2000) J. Neurochem., 74,

25. High resolving power (S20)
CalR
Rab11
CHC
1.22
1.175
1.130
1.085
1.04
g/ml
Endosomes
CCV
LER
DER
This long centrifugation time/low g-force strategy resolves endosomes, clathrin-coated vesicles (CCV) and two populations of endoplasmic reticulum (L, low-density and H, high-density) – only the latter contains paxillin and is thought to be perinuclear.
3T3 cell post-nuclear supernatant
10-40% iodixanol 48,000g/18 h
Woods, A.J. et al (2002) J. Biol. Chem., 277,

26. Publications database on subcellular membranes
OptiPrep (since 1994) approx 1200
Nycodenz® (since 1984) over 1000
Using either the Applications CD or the following website:
Follow the instructions to access the relevant Index
Click on the membrane of interest
Abstracts of all the papers reporting the use of OptiPrep or Nycodenz for subcellular membrane isolation and analysis can also be accessed from the same CD or website.