Isolation of Myelin

Jorge N. Larocca1, Williams T. Norton1

1 Albert Einstein College of Medicine, Yeshiva University, Bronx, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.25
DOI:  10.1002/0471143030.cb0325s33
Online Posting Date:  January, 2007
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The methods used to prepare myelin involve homogenization of the tissue in isotonic sucrose solution, followed by the isolation of myelin membranes by a series of steps that include density gradient centrifugation and differential centrifugation. Homogenization of nervous tissue in isotonic sucrose causes the myelin sheath to peel from the axon and form relatively large myelin vesicles. The large size of the myelin vesicles, together with the fact that myelin membrane has a lower density than other biological membranes, make differential centrifugation and density gradient centrifugation the main tools for the isolation of this membrane. Three protocols are outlined in this unit: isolation of a highly‐purified myelin fraction from the central nervous system (CNS); separation of a highly‐purified CNS myelin fraction into subfractions of different densities; and isolation of myelin from the peripheral nervous system (PNS).

Keywords: CNS myelin; PNS myelin; CNPase; oligodendrocytes; Schwann cell; brain; sciatic nerve

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Table of Contents

  • Basic Protocol 1: Isolation of Central Nervous System Myelin
  • Alternate Protocol 1: Separation of Highly Purified CNS Myelin Fraction Into Subfractions of Different Densities
  • Alternate Protocol 2: Isolation of PNS Myelin
  • Support Protocol 1: Assay for 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation of Central Nervous System Myelin

  • 200‐ to 300‐g Sprague‐Dawley rats (30 to 60 days old)
  • Anesthesia
  • 0.30 M and 0.83 M sucrose solutions (see recipe)
  • Tris⋅Cl buffer solution (see recipe)
  • Glass plate
  • Dissecting tools including: scissors, razor blades, spatulas, and forceps
  • 50‐ and 100‐ml beakers
  • Razor blades
  • 40‐ml Dounce homogenizer (Kontes) with both loose (clearance 0.0030 to 0.0060 in.) and tight (clearance 0.0010 to 0.0030 in.) pestles
  • 100‐ and 250‐ml graduated cylinder
  • 38.5‐ml thick‐walled ultracentrifuge tubes
  • Ultracentrifuge with swinging‐bucket rotor (Beckman SW 28, or equivalent)
  • Pasteur pipets
  • Glass rod
  • Additional reagents and equipment for determining protein concentration ( appendix 3H)

Alternate Protocol 1: Separation of Highly Purified CNS Myelin Fraction Into Subfractions of Different Densities

  • Highly‐purified myelin fraction (see protocol 1, step )
  • 0.30, 0.62, and 0.70 M sucrose solutions (see recipe)

Alternate Protocol 2: Isolation of PNS Myelin

  • 25‐ and 50‐ml beaker
  • 50‐ml Pyrex tube
  • Polytron homogenizer (Brinkmann Instruments)
  • Cheesecloth
  • Funnel

Support Protocol 1: Assay for 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase Activity

  • Myelin sample with 0.2 to 1 mg/ml protein
  • 0.2 M Tris⋅Cl buffer, pH 7.5 (see recipe)
  • 1% (w/v) sodium deoxycholate solution (see recipe)
  • CNPase reaction solution (see recipe)
  • 0.3 M Tris⋅Cl, pH 9.00, containing 21 mM MgCl 2
  • E. coli alkaline phosphatase solution (see recipe)
  • 1:1 (v/v) isobutanol/benzene
  • 1.5% (w/v) ammonium molybdate solution (see recipe)
  • 5‐ml Dounce homogenizer (Kontes) with tight (clearance 0.0010 to 0.0030 in.) pestle
  • Pyrex test tubes (10 × 75–mm)
  • 30° and 100°C water baths
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Literature Cited

Literature Cited
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