Purification of Organelles from Mammalian Cells

J. David Castle1

1 University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.1B
DOI:  10.1002/0471142735.im0801bs56
Online Posting Date:  November, 2003
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Abstract

Organelle purification procedures capitalize on the differences in size, density, and (occasionally) surface charge density of individual types of organelles. Most fractionation procedures that are based on centrifugation involve some combination of procedures that distinguish both size and density. Initially, a homogenate is prepared in isoosmotic (or slightly hyperosmotic) sucrose or some other predominantly nonelectrolyte medium. A wide range of procedures have been used to fractionate tissue homogenates. The protocols in this unit emphasize different fractionation techniques that have been used for rat liver, an abundant tissue that has been a favorite of many investigators and has served as the source of many organelle preparations of excellent purity. For selected procedures, examples have been given using other tissue sources (e.g., glandular tissues that maintain protein storage granules for regulated secretion) or, where particularly favorable, cultured cells.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Differential Centrifugation by Velocity
  • Basic Protocol 2: Differential Centrifugation by Equilibrium
  • Alternate Protocol 1: Rate Zonal Centrifugation Using Sucrose
  • Alternate Protocol 2: Gradient Fractionation Using Percoll
  • Alternate Protocol 3: Gradient Fractionation Using D2O
  • Alternate Protocol 4: Gradient Fractionation Using Nonionic Iodinated Solutes
  • Basic Protocol 3: Gel Filtration to Isolate Secretory Vesicles
  • Basic Protocol 4: Preparative Electrophoretic Separation
  • Alternate Protocol 5: Electrophoretic Separation on a Density Gradient
  • Basic Protocol 5: Solid‐Phase Immunoadsorption
  • Basic Protocol 6: Purification by Lectin Adsorption
  • Basic Protocol 7: Density Shift Using Digitonin
  • Basic Protocol 8: Density Shift Using Colloidal Gold Conjugates
  • Basic Protocol 9: Extraction of Extrinsic Proteins from Membranes Using Sodium Carbonate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Differential Centrifugation by Velocity

  Materials
  • Rat starved overnight and freshly sacrificed
  • Homogenization medium (see recipe)
  • Motor‐driven pestle with high‐torque motor (e.g., laboratory stirrer, VWR, or Fisher)
  • Cheesecloth
  • Tubes suitable for low‐speed centrifugation: 1.5‐ml microcentrifuge tubes (for small‐scale preparation) or 15‐ or 50‐ml graduated screw‐cap tubes (for larger‐scale preparations)
  • Low‐speed centrifuge capable of producing a 600 × g centrifugal field, 4°C
  • Teflon‐glass homogenizer (A.H. Thomas or equivalent) of appropriate size for the scale of the experiment: type AA (1 ml) up to type C (40 ml)
  • 200‐µm mesh nylon screen (Tetko)
  • Dounce‐type glass homogenizer
  • Polycarbonate or polyallomer tubes suitable for ultracentrifugation (e.g., 3‐ml size to fit Beckman TLA‐100.3 rotor) or polycarbonate bottle assemblies (e.g., 10‐ml size to fit Beckman 50Ti rotor or 25‐ml size to fit Beckman 60Ti or 70Ti rotor)
  • Ultracentrifuge and fixed‐angle rotor appropriate to the scale of the experiment (e.g., Beckman 50Ti, 60Ti, or 70Ti) or tabletop ultracentrifuge and rotor (e.g., Beckman tabletop with TL100 rotor)
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Basic Protocol 2: Differential Centrifugation by Equilibrium

  • Rat starved overnight and freshly sacrificed
  • Sucrose media: 0.25 M, 1.02 M, and 1.8 M sucrose (ultrapure; e.g., ICN Biochemicals) in dilution solution
  • Dilution solution: 50 mM Tris·Cl (pH 7.4)/25 mM KCl/5 mM MgCl 2
  • Ultraclear or polyallomer ultracentrifuge tubes to fit a swinging‐bucket rotor (e.g., 38 ml for Beckman SW28 rotor or equivalent; 13 ml for SW41 rotor or equivalent; 5 ml for SW50.1 or SW55 rotor or equivalent)
  • Gradient‐forming device
  • Ultracentrifuge and swinging‐bucket rotor (e.g., Beckman SW28, SW41, SW50.1, or SW55)
  • Syringe (with blunt needle), plastic transfer pipet, or siliconized glass Pasteur pipet
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Alternate Protocol 1: Rate Zonal Centrifugation Using Sucrose

  Materials
  • Rat starved overnight and freshly sacrificed
  • Homogenization medium: 0.3 M sucrose (ultrapure; e.g., ICN Biochemicals) plus 0.2 µg/ml diphenyl‐p‐phenylenediamine (DPPD, Kodak; from 0.4 mg/ml stock in ethanol)
  • 1 M MES
  • 0.2 M EDTA ( appendix 2A)
  • Gradient stock solutions, low‐ and high‐density (see recipe)
  • 0.3 M sucrose/5 mM MES/1 mM EDTA/0.2 µg/ml DPPD, pH 6.5 to 6.7
  • Cheesecloth
  • Dounce homogenizer
  • Gradient‐forming device suitable for selected gradient size, including compatible magnetic stir bars, tubing, and peristaltic pump
  • Ultraclear or polyallomer centrifuge tubes (of the selected size) suitable for swinging‐bucket rotor
  • Plastic tissue culture pipet or polyethylene transfer pipet
  • Ultracentrifuge and appropriate swinging‐bucket rotor (e.g., Beckman SW50.1 or SW55 if using 5‐ml tubes; Beckman SW41 for 12.5‐ml tubes; Beckman SW28 for 38‐ml tubes)
  • Automated gradient‐collection device (e.g., Buchler Autodensiflow, Nycomed Pharma or MSE Scientific) or micropipettor for manual collection
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Alternate Protocol 2: Gradient Fractionation Using Percoll

  • Percoll gradient solutions (see recipe)
  • Centrifuge and fixed‐angle rotor (e.g., Beckman 70Ti or Type 50)
  • Centrifuge tubes (e.g., Beckman thick‐walled polycarbonate bottle assemblies; 25‐ml for 70Ti rotor or 10‐ml for Type 50 rotor)
  • Refractometer
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Alternate Protocol 3: Gradient Fractionation Using D2O

  • Mouse pituitary AtT‐20/D16V cells
  • PBS ( appendix 2A) containing 4 mM EGTA (from 100 mM EGTA, pH 7.4), 37°C
  • Homogenization medium: 0.25 M sucrose (ultrapure; e.g., ICN Biochemicals)/10 mM HEPES (pH 7.4)/2 mM EGTA/1 mM EDTA, 4°C
  • Deuterium gradient stock solutions: 0.25 M ultrapure sucrose/10 mM HEPES (pH 7.4)/1 mM EDTA in D 2O (e.g., Aldrich), with and without 20% (w/v) Ficoll
  • 8% (w/v) Ficoll 70 (Pharmacia Biotech) in homogenization medium
  • 75‐cm2 tissue culture flasks
  • Dounce glass homogenizer with type B pestle (Kontes or equivalent)
  • Sorvall centrifuge and SS‐34 rotor (or equivalent)
  • Gradient‐forming device with capacity ≥15 ml/chamber (Hoefer Pharmacia or equivalent)
  • Peristaltic pump to form continuous gradient
  • Ultracentrifuge with swinging‐bucket rotor (e.g., Beckman SW27 or SW28)
  • Automated gradient collection apparatus: Autodensiflow device (Buchler) or gradient displacement device (Nycomed Pharma or MSE Scientific)
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after harvesting cells should be conducted at 0°C (ice bucket) or 4°C (cold room).

Alternate Protocol 4: Gradient Fractionation Using Nonionic Iodinated Solutes

  • Rat starved overnight and freshly sacrificed
  • Homogenization medium: 0.25 M sucrose (ultrapure; e.g., ICN Biochemicals), 4°C
  • 85.6% metrizamide (from 100% stock, Nycomed Pharma)
  • Gradient stock solutions: for continuous gradients, 19.7% and 50% (w/v) metrizamide, pH 7.4; for step gradients, 19.78%, 24.53%, 26.34%, and 32.82% (w/v) metrizamide, pH 7.4 (adjust pH with 0.01 N NaOH)
  • Teflon‐glass homogenizer (type C, 40 ml, A.H. Thomas)
  • Fixed‐angle ultracentrifuge rotor appropriate to the size of the experiment (e.g., Beckman 70Ti)
  • Automated gradient collection apparatus: Autodensiflow device (Buchler) or gradient displacement device (Nycomed Pharma or MSE Scientific)
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Basic Protocol 3: Gel Filtration to Isolate Secretory Vesicles

  Materials
  • Saccharomyces cerevisiae sec6‐mutant strain spheroplasted in 1.4 M sorbitol
  • Running buffer (see recipe)
  • 1.5 × 90–cm to 1.5 × 100–cm column packed with Sephacryl S‐1000 superfine resin (Pharmacia Biotech) and equilibrated with running buffer at 4°C
  • 1‐ml‐capacity Dounce homogenizer
  • Centrifuges: low‐speed and ultracentrifuge
  • Peristaltic pump capable of producing flow rates of ∼10 ml/hr
  • Fraction collector
  • Additional reagents and equipment for gel‐filtration chromatography (Hagel, )
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after harvesting cells should be conducted at 0°C (ice bucket) or 4°C (cold room).

Basic Protocol 4: Preparative Electrophoretic Separation

  Materials
  • Homogenization medium: 0.25 M sucrose (ultrapure; e.g., ICN Biochemicals)/50 mM 4‐morpholinoethanesulfonic acid (MES), pH 6.5
  • 6.25% sucrose/6.25% Ficoll 70/50 mM MES, pH 6.5
  • 50 mM MES, pH 6.5
  • Running buffer: 50 mM MES (pH 6.5) with added sucrose as desired
  • 0.15% to 0.2% Isogel agarose (FMC), or equivalent, in running buffer
  • Centrifuges: low‐speed and ultracentrifuge
  • Standard flatbed agarose gel electrophoresis apparatus with 0.06‐in. holes drilled in the cover to allow passage of inlet and outlet tubing from sample collecting wells (construct or purchase from Idea Scientific)
  • Standard 1.5‐mm‐thick plastic (siliconized) or Teflon gel comb
  • 1.5‐mm‐thick plastic (siliconized) or Teflon eluting combs cut so that teeth are 3 mm wider than loading wells
  • 0.062‐in.‐o.d. (1.57‐mm) polyethylene tubing (Clay‐Adams PE‐160)
  • Peristaltic pump capable of flow rates of 0.2 to 15.0 ml/hr
  • Fraction collector
  • Additional reagents and equipment for preparing microsomal fractions (see protocol 1) and analytical SDS‐PAGE (unit 8.7)

Alternate Protocol 5: Electrophoretic Separation on a Density Gradient

  • Rat starved overnight and freshly sacrificed
  • Electrophoresis buffer: 0.25 M sucrose (ultrapure; e.g., ICN Biochemicals)/10 mM triethanolamine/10 mM acetic acid/1 mM EDTA, pH 7.4
  • Trypsin, TPCK‐treated (Worthington Biochemical or equivalent)
  • Soybean trypsin inhibitor
  • 10%, 6%, 5%, and 4% (w/v) Ficoll 70, prepared in electrophoresis buffer
  • Electrophoresis apparatus: cylindrical electrophoresis chamber with dimensions of 5 cm high and 2.2 cm i.d. with circular palladium and platinum electrodes and cation‐permeable membrane (Ionics, Inc.; see Tulp et al., ) or Polyprep 2000 apparatus (Buchler)
  • Gradient‐forming device capable of forming linear concentration gradients of 10 ml volume (Hoefer Pharmacia)
  • 1‐mm‐gauge stainless steel mesh
  • Additional reagents and equipment for preparing microsomal fractions (see protocol 1)
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue, including forming and running the gradient, should be conducted at 4°C.

Basic Protocol 5: Solid‐Phase Immunoadsorption

  Materials
  • 5 mg/ml BSA in PBS
  • Primary antibody that selectively recognizes an antigenic epitope present on the exposed surface of the vesicle of interest
  • 5 mg/ml BSA in 0.25 M sucrose (ultrapure; e.g., ICN Biochemicals)/0.1 M potassium phosphate (pH 6.65)
  • Rat starved overnight and freshly sacrificed
  • Homogenization medium: 0.5 M sucrose/0.1 M potassium phosphate (pH 6.65)/5 mM MgCl 2
  • 1.25 M, 1.3 M, and 1.4 M sucrose (ultrapure) in 0.1 M potassium phosphate (pH 6.65)/5 mM MgCl 2
  • 4.5‐µm‐diameter magnetic beads coated with sheep anti‐mouse Fc antibody (Dynal)
  • Magnetic collection device (e.g., Dynal)
  • End‐over‐end rotator capable of slow rotation (2 to 4 rpm; e.g., Pelco rotator, modified)
  • 10‐ml and 38‐ml centrifuge tubes suitable, respectively, for Beckman Type 50 and SW28 rotors
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Basic Protocol 6: Purification by Lectin Adsorption

  Materials
  • Freshly sacrificed Sprague‐Dawley rat (200 to 275 g), fasted overnight
  • Homogenization medium (see recipe; add 0.1 mM PMSF just before fractionation), 0°C
  • 0.2 M KCl/0.2 M NaBr/50 mM Tris·Cl, pH 6.8
  • 50 mM Tris·Cl, pH 6.8 ( appendix 2A)
  • Wheat germ agglutinin (WGA) conjugated to Sepharose (Pharmacia Biotech), preequilibrated with 50 mM Tris·Cl, pH 6.8
  • 0.1 M N‐acetylglucosamine (e.g., Sigma) in 50 mM Tris·Cl, pH 6.8
  • Motor‐driven Teflon‐glass homogenizer
  • Cheesecloth
  • Centrifuge capable of producing a field of 25,000 × g and equipped with a fixed‐angle rotor
  • Centrifuge tubes
  • 5‐ml chromatography column
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Basic Protocol 7: Density Shift Using Digitonin

  Materials
  • Rat starved overnight to deplete glycogen stores and freshly sacrificed
  • Homogenization medium: 0.25 M sucrose (ultrapure; e.g., ICN Biochemicals)/3 mM imidazole (pH 7.4)
  • 20 mg/ml digitonin stock solution (see recipe)
  • 10% (w/v) Triton X‐100
  • Digitonin density shift gradient stock solutions (see recipe)
  • Sample for treatment and analysis
  • Motor‐driven Teflon‐glass homogenizer (type C, A.H. Thomas)
  • Cheesecloth
  • Low‐speed refrigerated centrifuge
  • Ultracentrifuge with fixed‐angle (e.g., Beckman 70Ti), and swinging‐bucket rotors (e.g., Beckman SW28)
  • Ultracentrifuge tubes to fit rotors
  • Gradient‐forming device (e.g., Hoefer, Pharmacia)
  • Additional reagents and equipment for microsomal fraction preparation (see protocol 1)
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).

Basic Protocol 8: Density Shift Using Colloidal Gold Conjugates

  Materials
  • 1% (w/v) trisodium citrate dihydrate
  • 1% (w/v) tannic acid (Mallinkrodt)
  • 25 mM K 2CO 3
  • 1% (w/v) chloroauric acid (HAuCl 4; Fisher) in twice‐distilled water (store in brown bottle; stable for months at 4°C)
  • Proteins for conjugation: ricin (galactose‐specific lectin; Sigma), anti‐mouse transferrin receptor antibody (I.W. Trowbridge), and low‐density lipoprotein (LDL; density 1.019 to 1.063 g/ml)
  • 10% (w/v) NaCl
  • 1% (w/v) BSA
  • Phosphate‐buffered saline (PBS, appendix 2A) containing 0.5% polyethylene glycol (PEG) 20,000 (Carbowax 20M, Fluka)
  • Cultured cells of an appropriate cell line to be used as a source of membrane fractions: e.g., BW 5147 mouse T cell lymphoma cells (characterized by I.W. Trowbridge, Salk Institute; available from ATCC) or HEp.2 cells (ATCC CCL23)
  • Dulbeccos Modified Eagle Medium (DMEM) containing 100 mg/ml BSA
  • DMEM containing 1% horse serum
  • DMEM containing 5 mg/ml BSA
  • PBS ( appendix 2A)
  • Hypoosmotic homogenization medium (see recipe)
  • Hyperosmotic buffer (see recipe)
  • RNase (Sigma)
  • DNase I for treating cell homogenates (Sigma)
  • Pellet resuspension solution: 0.25 M sucrose (ultrapure; e.g., ICN Biochemicals)/1 mM EDTA/10 mM Tris acetate (pH 8.0)
  • Continuous density gradient solutions: 30% and 60% (w/v) sucrose (ultrapure) in 1 mM EDTA/10 mM Tris acetate (pH 7.5)
  • 2.5 M sucrose in water
  • Nitrogen bomb and Dounce homogenizer for homogenization
  • Low‐speed refrigerated centrifuge
  • Ultracentrifuge with fixed‐angle and swinging‐bucket rotors (e.g., Beckman ultracentrifuge with Type 50 and SW40 or SW41 rotors)
  • Gradient‐forming device
  • Micropipettor
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after harvesting cells should be conducted at 0°C (ice bucket) or 4°C (cold room).

Basic Protocol 9: Extraction of Extrinsic Proteins from Membranes Using Sodium Carbonate

  Materials
  • 0.1 M sodium carbonate (Na 2CO 3), pH 11.5, ice cold
  • 0.3 M sucrose (ultrapure; e.g., ICN Biochemicals), buffered to pH 7 to 7.5 (e.g., with 10 mM Tris·Cl)
  • Cell organelle or membrane fraction to be treated
  • Ultracentrifuge with swinging‐bucket rotor
NOTE: Prepare solutions with Milli‐Q‐purified water or equivalent.NOTE: All operations after removing and weighing tissue should be conducted at 0°C (ice bucket) or 4°C (cold room).
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Literature Cited

Literature Cited
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