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Fractionation of organelles and membrane vesicles using
OptiPrep™
• Competitive products are sucrose and Percoll®
• Sucrose-based publications go back to 1948
Plasma membrane
Lipid raftsCaveolae
Coated pits
Exocytosis/secretion
Endocytosis
N
M LP
Endoplasmic reticulum
Golgi
Trans-Golgi network
Endocyticcompartments
ERGIC
Iodixanol gradient solution strategy (S01, S02)
• Simple gradient solution preparation• When OptiPrep is diluted with the
homogenization medium the solutions will be iso-osmotic
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
Homogenize cells in 0.25 M sucrose,1 mM EDTA, 10 mM Tris-HCl, pH 7.4
1000g/10 min
pelletnuclei
supernatantsupernatant
15,000g/15 min
100,000g/60 minPellet (LMF)mitochondria
lysosomesperoxisomes
pelletvesicles (microsomes)
supernatant cytosol
Differential centrifugation of homogenate
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
Analyze fractions
• 20-30 fractions (0.5-1.0 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
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
• 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
Density gradient medium should not interfere with analysis II
Four important examples of simple Four important examples of simple discontinuous iodixanol gradientsdiscontinuous 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
Purification of nuclei I (S08)
10,000g20 min
homogenate in25% iodixanol
30% iodixanol
35% iodixanol
nuclei
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 (100-150,000g 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
Density of particles in iodixanol allows superior resolution
Organelle Sucrose Iodixanol
Mitochondria 1.17-1.21 1.13-1.16
Lysosomes 1.19-1.21 1.11-1.13
Peroxisomes 1.18-1.23 1.17-1.21
Nuclei >1.32 1.23-1.25
Purification of mitochondria (S12)Purification of mitochondria (S12)
50,000g4h
Crude mitochondrialpellet (1.204 g/ml)
1.079 g/ml
1.175 g/ml
Mitochondria
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
Isolation of peroxisomes II%
Dis
tribu
tion
Den
sity
(g/m
l)
1 3 5 7 9 11 13 15 17 190
10
20
30
40
1.05
1.1
1.15
1.2
1.25
1.3Density
Glut deHase
Catalase
Acid Pase
G-6-Pase
63%
LMF in self-generated gradient (S14)LMF in self-generated gradient (S14)
Fraction Number
% D
istri
butio
n
Den
sity
(g/m
l)
1 3 5 7 9 11 13 15 170
10
20
30
40
50
60
1.06
1.08
1.1
1.12
1.14
1.16
1.18
1.2Density Succ deHase Catalaseß-Gal'ase Gal trans
Vesicle/cytosol separation (S36)Vesicle/cytosol separation (S36)
200-300,000g1-3 h
Crude vesiclefraction 1.16 g/ml
1.05 g/ml
1.14 g/mlVesicles
Cytosolicproteins
Lipid rafts
Endoplasmic reticulumPlasma membrane
Caveolae
Coated pits
Exocytosis/secretion
Endocytosis
N
M LP
Golgi
Trans-Golgi network
Endocyticcompartments
ERGIC
Isolation of detergent-insoluble membranes (S33)
Iodixanol conc.
20%
35%
40%
HM
PNS
160,000g
4 h
Lipid rafts
Dissection of lipid-rich domains in Dissection 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
Plasma membraneEndoplasmic reticulum
Golgi
Trans-Golgi networkLipid raftsCaveolae
Coated pits
Exocytosis/secretion
Endocytosis
N
M LP Endocyticcompartments
ERGIC
Fraction number
% M
axim
um
Den
sity
(g/m
l)
1 3 5 7 9 11 13 15 17 190
20
40
60
80
100
1
1.02
1.04
1.06
1.08
1.1
1.12
From Yang, M et al (1997) J. Biol. Chem., 272, 1970-1975CHO cell PNS on 0-26% iodixanol gradient: 200,000g for 2h
ERGolgi
PM
High resolving power (S19)High resolving power (S19)
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, 1878-1884
Calnexin
ßCOP
Rab8
Fraction # 1 3 5 7 9
High resolving power (S23)High resolving power (S23)
CalR
Rab11
CHC
1.22
1.175
1.130
1.085
1.04
g/ml
Endosomes CCV LER DER
3T3 cell post-nuclear supernatant10-40% iodixanol 48,000g/18 hWoods, A.J. et al (2002) J. Biol. Chem., 277, 6428-6437
High resolving power (S20)High resolving power (S20)
Publications database on Publications database on subcellular membranes subcellular membranes
• OptiPrep (since 1994) approx 1200• Nycodenz® (since 1984) over 1000• Using either the Applications CD or the following
website:• www.axis-shield-density-gradient-media.com• Follow the instructions to access the relevant
Index• Click on the membrane of interest
