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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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Presentation on theme: "Fractionation of organelles and membrane vesicles using OptiPrep™ Competitive products are sucrose and Percoll® Sucrose-based publications go back to 1948."— Presentation transcript:

1 Fractionation of organelles and membrane vesicles using OptiPrep™ Competitive products are sucrose and Percoll® Sucrose-based publications go back to 1948

2 Plasma membrane Lipid rafts Caveolae Coated pits Exocytosis/secretion Endocytosis N M L P Endoplasmic reticulum Golgi Trans-Golgi network Endocytic compartments ERGIC

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

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

5 Homogenize cells in 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH g/10 min pellet nuclei supernatant 15,000g/15 min 100,000g/60 min Pellet (LMF) mitochondria lysosomes peroxisomes pellet vesicles (microsomes) supernatant cytosol Differential centrifugation of homogenate

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

7 Analyze fractions 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

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

9 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

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

11 Purification of nuclei I (S08) 10,000g 20 min homogenate in 25% iodixanol 30% iodixanol 35% iodixanol nuclei

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

13 Density of particles in iodixanol allows superior resolution OrganelleSucroseIodixanol Mitochondria Lysosomes Peroxisomes Nuclei>

14 Purification of mitochondria (S12) 50,000g 4h Crude mitochondrial pellet (1.204 g/ml) g/ml g/ml 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

16 Isolation of peroxisomes II

17 LMF in self-generated gradient (S14)

18 Vesicle/cytosol separation (S36) ,000g 1-3 h Crude vesicle fraction  1.16 g/ml  1.05 g/ml  1.14 g/ml Vesicles Cytosolic proteins

19 Lipid rafts Endoplasmic reticulum Plasma membrane Caveolae Coated pits Exocytosis/secretion Endocytosis N M L P Golgi Trans-Golgi network Endocytic compartments ERGIC

20 Isolation of detergent-insoluble membranes (S33) Iodixanol conc. 20% 35% 40% HM PNS 160,000g 4 h Lipid rafts

21 Dissection of lipid-rich domains in iodixanol gradients Dissection of lipid-rich domains in iodixanol gradients Adapted from Lindwasser, OW and Resh MD (2001) J. Virol., 75, % 40% 50% 30% 20% Caveolin Cholesterol GM1 Na + /K + -ATPase

22 Plasma membrane Endoplasmic reticulum Golgi Trans-Golgi network Lipid rafts Caveolae Coated pits Exocytosis/secretion Endocytosis N M L P Endocytic compartments ERGIC

23 Fraction number % Maximum Density (g/ml) From Yang, M et al (1997) J. Biol. Chem., 272, CHO cell PNS on 0-26% iodixanol gradient: 200,000g for 2h ER Golgi PM High resolving power (S19)

24 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, Calnexin ßCOP Rab8 Fraction # High resolving power (S23)

25 CalR Rab11 CHC g/ml EndosomesCCVLERDER 3T3 cell post-nuclear supernatant 10-40% iodixanol 48,000g/18 h Woods, A.J. et al (2002) J. Biol. Chem., 277, High resolving power (S20)

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


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