Isolation of Intermediate Filaments

Conrad L. Leung1, Ronald K.H. Liem1

1 Columbia University, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.23
DOI:  10.1002/0471143030.cb0323s31
Online Posting Date:  July, 2006
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Abstract

Intermediate filaments (IFs) are found in most eukaryotic cells and are made up of various IF proteins. IFs are highly insoluble in conventional extraction buffers and are therefore commonly purified under denaturing condition. Purified IF proteins can be reassembled into filaments by dialysis. At least 65 IF proteins are found in humans, and the procedures for the purification of each subunit vary somewhat, although many basic steps are similar. To illustrate the isolation of IFs, a detailed protocol is described for purifying neurofilament proteins (NFL, NFM, and NFH subunits) from bovine spinal cord. These three proteins form the predominant IF network in mature neurons. An alternative method for the purification of NFL from a prokaryotic expression system is also included. The isolation of recombinant proteins from bacteria is quite straightforward and may therefore be the method of choice for producing and purifying IFs. Finally, there is a discussion of the purification methods of other IF proteins.

Keywords: Intermediate filaments; neurofilaments; cytoskeleton; recombinant proteins

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Neurofilaments from Bovine Spinal Cord
  • Alternate Protocol 1: Isolation of Recombinant Neurofilament Light Subunit (NFL)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Neurofilaments from Bovine Spinal Cord

  Materials
  • Fresh bovine spinal cord (can be obtained from a local slaughterhouse)
  • Solution A (see recipe), ice cold
  • Solution A‐TSP (see recipe), ice cold
  • Buffers 1, 2, 3, and 4 (see reciperecipes)
  • Bio‐Gel HTP resin (Bio‐Rad) or equivalent hydroxylapatite resin
  • 10 mM sodium phosphate buffer, pH 7.4 ( appendix 2A), degassed under vacuum
  • Assembly buffer (see recipe)
  • 7.5% SDS‐PAGE gel (unit 6.1)
  • DEAE‐cellulose resin (Sigma or Whatman)
  • Buffer 4 (see recipe) containing 1% (v/v) 2‐mercaptoethanol
  • Buffer 4 (see recipe), pH 6.5, containing 55 mM NaCl
  • Buffer 4 (see recipe), pH 7.0, containing 66 mM NaCl
  • Dounce homogenizer
  • Refrigerated centrifuge and centrifuge bottles
  • 1.5 × 10–cm glass barrel chromatography column (Bio‐Rad)
  • Peristaltic pump and fraction collector
  • 0.45‐µm syringe filters
  • Amicon Ultra‐15 centrifugal filter units, 30‐kDa MWCO (Millipore), or equivalent
  • Dialysis cassette, 10‐kDa MWCO
  • Ultracentrifuge
  • Additional reagents and equipment for spectrophotometric determination of protein ( appendix 3B) and SDS‐PAGE (unit 6.1)

Alternate Protocol 1: Isolation of Recombinant Neurofilament Light Subunit (NFL)

  Materials
  • NFL cDNA: generated by reverse‐transcription PCR using RNA from brain of mouse (or other species) or obtained as EST clone from I.M.A.G.E. Consortium (http://image.llnl.gov/)
  • pET16d vector (Novagen) or equivalent
  • BL21(DE3) E. coli competent cells or equivalent
  • LB‐ampicillin plates and LB‐ampicillin liquid medium ( appendix 2A; see appendix 3A for cross‐references to selection methods)
  • 100 mM isopropyl‐β‐D‐thiogalactopyranoside (IPTG; see appendix 3A for cross‐references to protein expression methods)
  • 2× SDS sample buffer ( appendix 2A)
  • 20 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • Buffer, 1, 2, and 3 (see reciperecipes)
  • Refrigerated centrifuge
  • Probe sonicator
  • Additional reagents and equipment for molecular biology techniques (cloning, restriction digestion, transformation of E. coli, selection of transformants and growth in LB‐ampicillin media, induction of expression with IPTG; see appendix 3A), and SDS‐PAGE (unit 6.1)
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Figures

Videos

Literature Cited

Literature Cited
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