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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


Basic Protocol 1: Isolation of Neurofilaments from Bovine Spinal Cord

  • 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)

  • 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 (
  • 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)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Bilak, S.R., Sernett, S.W., Bilak, M.M., Bellin, R.M., Stromer, M.H., Huiatt, T.W., and Robson, R.M. 1998. Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle. Arch. Biochem. Biophys. 355:63‐76.
   Chou, C.F., Riopel, C.L., Rott, L.S., and Omary, M.B. 1993. A significant soluble keratin fraction in ‘simple’ epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility. J. Cell Sci. 105:433‐444.
   Coulombe, P.A. and Fuchs, E. 1990. Elucidating the early stages of keratin filament assembly. J. Cell Biol. 111:153‐169.
   Eriksson, J.E., He, T., Trejo‐Skalli, A.V., Harmala‐Brasken, A.S., Hellman, J., Chou, Y.H., and Goldman, R.D. 2004. Specific in vivo phosphorylation sites determine the assembly dynamics of vimentin intermediate filaments. J. Cell Sci. 117:919‐932.
   Fradette, J., Germain, L., Seshaiah, P., and Coulombe, P.A. 1998. The type I keratin 19 possesses distinct and context‐dependent assembly properties. J. Biol. Chem. 273:35176‐35184.
   Geisler, N. and Weber, K. 1980. Purification of smooth‐muscle desmin and a protein‐chemical comparison of desmins from chicken gizzard and hog stomach. Eur. J. Biochem. 111:425‐433.
   Geisler, N. and Weber, K. 1981. Isolation of polymerization‐competent vimentin from porcine eye lens tissue. FEBS Lett. 125:253‐256.
   Goulielmos, G., Gounari, F., Remington, S., Muller, S., Haner, M., Aebi, U., and Georgatos, S.D. 1996. Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells. J. Cell Biol. 132:643‐655.
   Heitlinger, E., Peter, M., Haner, M., Lustig, A., Aebi, U., and Nigg, E.A. 1991. Expression of chicken lamin B2 in Escherichia coli: Characterization of its structure, assembly, and molecular interactions. J. Cell Biol. 113:485‐495.
   Hemken, P.M., Bellin, R.M., Sernett, S.W., Becker, B., Huiatt, T.W., and Robson, R.M. 1997. Molecular characteristics of the novel intermediate filament protein paranemin: Sequence reveals EAP‐300 and IFAPa‐400 are highly homologous to paranemin. J. Biol. Chem. 272:32489‐32499.
   Herrmann, H., Haner, M., Brettel, M., Ku, N.O., and Aebi, U. 1999. Characterization of distinct early assembly units of different intermediate filament proteins. J. Mol. Biol. 286:1403‐1420.
   Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., and Sato, C. 1987. Site‐specific phosphorylation induces disassembly of vimentin filaments in vitro. Nature 328:649‐652.
   Liem, R.K. 1986. Purification of neurofilament and their constituent polypeptides. In The Contractile Apparatus and the Cytoskeleton, Vol. 134 (R.B. Vallee ed.) pp. 380‐387. Elsevier Academic Press, Orlando, Fla.
   Liem, R.K. and Hutchison, S.B. 1982. Purification of individual components of the neurofilament triplet: Filament assembly from the 70,000‐dalton subunit. Biochemistry 21:3221‐3226.
   Newey, S.E., Howman, E.V., Ponting, C.P., Benson, M.A., Nawrotzki, R., Loh, N.Y., Davies, K.E., and Blake, D.J. 2001. Syncoilin, a novel member of the intermediate filament superfamily that interacts with alpha‐dystrobrevin in skeletal muscle. J. Biol. Chem. 276:6645‐6655.
   O'Shea, J.M., Robson, R.M., Hartzer, M.K., Huiatt, T.W., Rathbun, W.E., and Stromer, M.H. 1981. Purification of desmin from adult mammalian skeletal muscle. Biochem. J. 195:345‐356.
   Omary, B. and Coulombe, P.A. 2004. Intermediate Filament Cytoskeleton. Methods in Cell Biology, Vol. 78 (L. Wilson and P. Matsundaira, eds.). Elsevier Academic Press, San Diego, Calif.
   Pachter, J.S. and Liem, R.K. 1985. Alpha‐Internexin, a 66‐kD intermediate filament‐binding protein from mammalian central nervous tissues. J. Cell Biol. 101:1316‐1322.
   Parry, D.A. and Steinert, P.M. 1995. Intermediate Filament Structure. R.G. Landes, Austin, Tx.
   Parysek, L.M. and Goldman, R.D. 1987. Characterization of intermediate filaments in PC12 cells. J. Neurosci. 7:781‐791.
   Poon, E., Howman, E.V., Newey, S.E., and Davies, K.E. 2002. Association of syncoilin and desmin: Linking intermediate filament proteins to the dystrophin‐associated protein complex. J. Biol. Chem. 277:3433‐3439.
   Pytela, R. and Wiche, G. 1980. High molecular weight polypeptides (270,000‐340,000) from cultured cells are related to hog brain microtubule‐associated proteins but copurify with intermediate filaments. Proc. Natl. Acad. Sci. U.S.A. 77:4808‐4812.
   Quinlan, R.A., Carter, J.M., Hutcheson, A.M., and Campbell, D.G. 1992. The 53 kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115 kDa beaded filament protein: Evidence for a fibre cell specific intermediate filament protein. Curr. Eye Res. 11:909‐921.
   Rueger, D.C., Huston, J.S., Dahl, D., and Bignami, A. 1979. Formation of 100 Å filaments from purified glial fibrillary acidic protein in vitro. J. Mol. Biol. 135:53‐68.
   Sandoval, I.V., Colaco, C.A., and Lazarides, E. 1983. Purification of the intermediate filament‐associated protein, synemin, from chicken smooth muscle: Studies on its physicochemical properties, interaction with desmin, and phosphorylation. J. Biol. Chem. 258:2568‐2576.
   Steinert, P.M., Idler, W.W., Cabral, F., Gottesman, M.M., and Goldman, R.D. 1981. In vitro assembly of homopolymer and copolymer filaments from intermediate filament subunits of muscle and fibroblastic cells. Proc. Natl. Acad. Sci. U.S.A. 78: 3692‐3696.
   Steinert, P.M., Chou, Y.H., Prahlad, V., Parry, D.A., Marekov, L.N., Wu, K.C., Jang, S.I., and Goldman, R.D. 1999. A high molecular weight intermediate filament‐associated protein in BHK‐21 cells is nestin, a type VI intermediate filament protein: Limited co‐assembly in vitro to form heteropolymers with type III vimentin and type IV alpha‐internexin. J. Biol. Chem. 274:9881‐9890.
PDF or HTML at Wiley Online Library