fractionation of organelles and membrane vesicles using optiprep™ competitive products are sucrose...
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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
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Plasma membrane
Lipid rafts
Caveolae
Coated pits
Exocytosis/secretion
Endocytosis
N
M LP
Endoplasmic reticulum
Golgi
Trans-Golgi network
Endocyticcompartments
ERGIC
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Iodixanol gradient solution strategy (S01, S02)
• Simple gradient solution preparation
• When OptiPrep is diluted with the homogenization medium the solutions will be iso-osmotic
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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
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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
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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
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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
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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
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• 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
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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
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Purification of nuclei I (S08)
10,000g
20 min
homogenate in25% iodixanol
30% iodixanol
35% iodixanol
nuclei
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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
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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
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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
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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
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Isolation of peroxisomes II%
Dis
trib
utio
n
Den
sity
(g/
ml)
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%
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LMF in self-generated gradient (S14)LMF in self-generated gradient (S14)
Fraction Number
% D
istr
ibut
ion
Den
sity
(g/
ml)
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
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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
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Lipid rafts
Endoplasmic reticulumPlasma membrane
Caveolae
Coated pits
Exocytosis/secretion
Endocytosis
N
M LP
Golgi
Trans-Golgi network
Endocyticcompartments
ERGIC
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Isolation of detergent-insoluble membranes (S33)
Iodixanol conc.
20%
35%
40%
HM
PNS
160,000g
4 h
Lipid rafts
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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
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Plasma membraneEndoplasmic reticulum
Golgi
Trans-Golgi networkLipid rafts
Caveolae
Coated pits
Exocytosis/secretion
Endocytosis
N
M LP Endocyticcompartments
ERGIC
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Fraction number
% M
axim
um
Den
sity
(g/
ml)
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
ER
Golgi
PM
High resolving power (S19)High resolving power (S19)
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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)
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CalR
Rab11
CHC
1.22
1.175
1.130
1.085
1.04
g/ml
Endosomes CCV LER DER
3T3 cell post-nuclear supernatant
10-40% iodixanol 48,000g/18 h
Woods, A.J. et al (2002) J. Biol. Chem., 277, 6428-6437
High resolving power (S20)High resolving power (S20)
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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