Fractionation of organelles and membrane vesicles using

OptiPrep™

• Competitive products are sucrose and Percoll®

• Sucrose-based publications go back to 1948

Plasma membrane

Lipid rafts

Caveolae

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)

10,000g

20 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

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%

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

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 rafts

Caveolae

Coated pits

Exocytosis/secretion

Endocytosis

N

M LP Endocyticcompartments

ERGIC

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)

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 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)

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