Isolation of Microtubules and Microtubule Proteins

J. Avila1, H. Soares2, M.L. Fanarraga3, J.C. Zabala3

1 Centro de Biología Molecular (CSIC). Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain, 2 Instituto Gulbenkian de Ciência, 2781‐901, Oeiras, and Escola Superior de Tecnologia da Saúde de Lisboa, Lisboa, Portugal, 3 Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria‐ IFIMAV, Santander, Spain
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.29
DOI:  10.1002/0471143030.cb0329s39
Online Posting Date:  June, 2008
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Abstract

This unit describes various protocols for the isolation and purification of the main constituents of microtubules, chiefly α‐ and β‐tubulin, and the most significant microtubule associated proteins (MAPs), specifically MAP1A, MAP1B, MAP2, and tau. We include a classical isolation method for soluble tubulin heterodimer as the first basic purification protocol. In addition, we show how to analyze the tubulin and MAPs obtained after a phosphocellulose chromatography purification procedure. This unit also details a powerful and simple method to determine the native state of the purified tubulin based on one‐dimensional electrophoresis under nondenaturing conditions (UNIT 6.5). The last protocol describes the application of a new technique that allows visualizing the quality of polymerized microtubules based on atomic force microscopy (AFM). Curr. Protoc. Cell Biol. 39:3.29.1‐3.29.28. © 2008 by John Wiley & Sons, Inc.

Keywords: tubulin; microtubule; MAP1A; MAP1B; MAP2; tau; native gel electrophoresis; atomic force microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Microtubule Protein by Cycles of Polymerization and Depolymerization
  • Basic Protocol 2: Isolation of Microtubule Protein Using Paclitaxel (Taxol)
  • Alternate Protocol 1: Microtubule Polymerization in the Presence of Paclitaxel
  • Basic Protocol 3: Isolation of Tubulin Dimers from Microtubule Proteins
  • Basic Protocol 4: Isolation of MAP1A/1B
  • Basic Protocol 5: Isolation of High‐Molecular‐Weight MAP2
  • Basic Protocol 6: Isolation of Tau Protein
  • Support Protocol 1: Nondenaturing Polyacrylamide Gel Electrophoresis as a Simple Method to Quantify/Check Polymerization‐Competent Tubulin
  • Support Protocol 2: Visualization of Polymerized Microtubules by Atomic Force Microscopy (AFM)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Microtubule Protein by Cycles of Polymerization and Depolymerization

  Materials
  • One to three brains (100 to 500 g total start‐up tissue; the weight of a pig brain is ∼100 g)
  • Phosphate‐buffered saline, pH 6.7 (PBS; appendix 2A), 4°C
  • Isotonic buffer (see recipe), 4°C
  • Protease inhibitors (see recipe)
  • Buffer A (see recipe), 4°C
  • Glycerol
  • PMSF
  • GTP (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Plastic wrap
  • Ice‐water bath
  • No.10 scalpel blade
  • 500‐ml beaker
  • Potter homogenizer (Teflon‐in‐glass homogenizer), as large as possible
  • Centrifuge (if possible two centrifuges, one at 4° to 6°C and the other at 25° to 30°C): Sorvall RC‐5B or equivalent Beckman J2, J21 series, or Avanti J‐25
  • Centrifuge rotors: GSA or JA‐10; type 50.2Ti and 70.1 Ti for the Beckman
  • Graduated flask
  • 26.3‐ml ultracentrifuge tubes
  • 35°C rocking incubator
  • Ultracentrifuge Beckman L8‐70, Optima XL‐100 or 120
  • Additional reagents and equipment for measuring protein concentration ( appendix 3B)

Basic Protocol 2: Isolation of Microtubule Protein Using Paclitaxel (Taxol)

  Materials
  • Bovine or porcine brain tissue (10 g)
  • Phosphate‐buffered saline ( appendix 2A)
  • Buffer A (see recipe)
  • Protease inhibitors (see recipe)
  • Paclitaxel (Taxol; see recipe)
  • GTP (see recipe)
  • PMSF
  • Sucrose underlayer solution (1 ml per centrifuge tube containing 10% sucrose, 10 µM paclitaxel, and 0.5 mM GTP)
  • Scalpel blade, No.10
  • 500‐ml beaker
  • Potter homogenizer (Teflon‐in‐glass homogenizer)
  • Centrifuge rotors: GSA or JA‐10. Type 50.2Ti and 70.1 Ti for the Beckman
  • Ultracentrifuge Beckman L8‐70, Optima XL‐100 or 120
  • 13.5‐ml ultracentrifuge tubes
  • 30°C rocking incubator
  • Pasteur pipet

Alternate Protocol 1: Microtubule Polymerization in the Presence of Paclitaxel

  Materials
  • Resin: Whatman P11 Cellulose Phosphate
  • 0.5 M NaOH
  • 0.5 M HCl
  • Buffer A (see recipe)
  • 50 mg of porcine brain microtubules stored at −80°C or in liquid nitrogen ( protocol 1)
  • Buchner funnel
  • XK50/20 (Length 20 cm, i.d. 50 mm; Pharmacia) column including:
    • Thermostat jacket
    • Flow adaptor
    • Flanged tubing at both ends for direct connection to valves
    • Pumps
    • UV monitors if using an FPLC or an AKTA system
  • Teflon‐in‐glass homogenizer
  • Ice‐water bath
  • Ultracentrifuge
  • Additional reagents and equipment for isolating microtubule proteins ( protocol 1), determining protein concentration ( appendix 3B), SDS‐PAGE (unit 6.1), and nondenaturing gel electrophoresis ( protocol 8)

Basic Protocol 3: Isolation of Tubulin Dimers from Microtubule Proteins

  Materials
  • White matter from a cow or ten brains from adults rats (MAP1A)
  • Twenty brains from newborns rats (MAP1B)
  • Buffer B (see recipe)
  • GTP
  • Paclitaxel (Taxol; see recipe)
  • Poly‐L‐aspartic
  • Buffer C (see recipe)
  • Buffer D (see recipe)
  • Buffer A (see recipe)
  • 10.4‐ml centrifuge tubes
  • Ice‐water bath
  • Centrifuge
  • Centrifuge rotors: GSA or JA‐10. Type 50.2Ti and 70.1 Ti for the Beckman
  • 100‐ml Erlenmeyer flask
  • 37°C incubator
  • Ultracentrifuge Beckman L8‐70, Optima XL‐100 or 120
  • 1‐ml Mono‐Q column (or another with similar characteristics) connected to a FPLC or AKTA system
  • Additional reagents and equipment for preparing and homogenizing the brains ( protocol 1), FPLC (Pazzagli and Avila, ), and SDS‐PAGE (unit 6.1)

Basic Protocol 4: Isolation of MAP1A/1B

  Materials
  • Porcine or bovine brains or alternatively microtubule protein stored at −80°C or liquid nitrogen ( protocol 1)
  • Buffer A (see recipe)
  • NaCl
  • 2‐mercaptoethanol
  • Ammonium sulfate
  • Sepharose 4B resin (GE Healthcare‐Pharmacia)
  • Teflon‐glass homogenizer
  • Ice‐water bath
  • Centrifuge
  • Centrifuge rotors: GSA or JA‐10, Type 50.2 Ti and 70.1 Ti for the Beckman
  • Boiling water bath
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1)

Basic Protocol 5: Isolation of High‐Molecular‐Weight MAP2

  Materials
  • Porcine brain
  • Buffer A (see recipe)
  • Perchloric acid
  • Ammonium sulfate
  • Glycerol
  • Ice‐water bath
  • Boling water bath
  • Centrifuge
  • Centrifuge rotors: GSA or JA‐10. Type 50.2Ti and 70.1 Ti for the Beckman

Basic Protocol 6: Isolation of Tau Protein

  Materials
  • Gel solutions (Table 3.29.1)
    Table 3.9.1   Materials   Recipe for Gel Preparation Using a Native Continuous Buffer System a   Recipe for Gel Preparation Using a Native Continuous Buffer System

    Final acrylamide concentration in gel (%) c
    Stock solution (ml) b 4 5 6 7
    Distilled water 6.48 6.15 5.81 5.48
    30% acrylamide/0.8% bisacrylamide d 1.33 1.66 2 2.33
    0.5 M MES, pH 6.7 2 2 2 2
    1 M MgCl 2 0.01 0.01 0.01 0.01
    0.5 M EGTA 0.02 0.02 0.02 0.02
    1 M GTP 0.01 0.01 0.01 0.01
    10% (w/v) APS (ammonium persulfate) e 0.14 0.14 0.14 0.14
    TEMED f 0.01 0.01 0.01 0.01

     aPreparation of the gel: Use a 75 or 100‐ml Erlenmeyer flask or in order to speed polymerization, degas the mix under vacuum several minutes in a side‐arm flask. Mix the ProtoGelc solution with 0.5M MES pH 6.7, 1M MgCl 2, 1M GTP and 0.5M EGTA. Add 10% (w/v) ammonium persulfate and TEMED.
     bAll reagents and solutions used must be prepared with Milli‐Q purified water or equivalent.
     cThe recipe is for 10 ml solution, which is adequate for 2 minigels (7 cm × 8 cm) 0.75 mm thickness.
     dThe polyacrylamide can be prepared as described in Chapter 6, but it is more convenient to use a ready‐to‐use commercial solution, we usually use ProtoGel 30% (w/v) acrylamide:0.8% (w/v) bis‐acrylamide from National Diagnostics, England.
     eIt can be prepared and stored frozen at −20°C in aliquots but we prefer to use freshly made.
     fAdd just before polymerization.
  • 4× loading buffer [50 mM MES, pH 6.7, containing 30%(v/v) glycerol or 25%(w/v) sucrose
  • Electrophoresis (running) buffer (see recipe)
  • GTP
  • Protein sample to be analyzed
  • 10× transfer buffer (see recipe), optional
  • Electrophoresis equipment (Chapter 6; e.g., Miniprotean from Bio‐Rad and 0.75‐mm spacers)
  • Additional reagents and equipment for gel staining (unit 6.2)

Support Protocol 1: Nondenaturing Polyacrylamide Gel Electrophoresis as a Simple Method to Quantify/Check Polymerization‐Competent Tubulin

  Materials
  • Mica (muscovite; SPI Supplies)
  • Nail polish or water‐resistant glue (e.g., epoxy resin)
  • Silanization solution: 0.1% (v/v) 3‐aminopropyl‐triethoxysilane (APTES; Fluka, 96%) freshly prepared with Milli‐Q water
  • Milli‐Q water; if unavailable, bidistilled water filtered through a 0.22‐µm pore filter
  • GTP
  • Paclitaxel (taxol)
  • Sample of purified tubulin ( protocol 4)
  • 0.1 M PIPES, pH 6.9
  • 1 mM EGTA
  • 1 mM MgCl 2
  • 1 mM PMSF
  • Sharp scissors
  • Glass coverslips or slides
  • Duct tape
  • Micropipet
  • 30°C thermostatic bath
  • AFM Si tapping mode tips (Olympus, OTESP)
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Figures

Videos

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Internet Resources
   http://mitchison.med.harvard.edu/protocols/tubprep.html
  Web site for large scale tubulin preparation protocol.
   http://mitchison.med.harvard.edu/protocols/recycle.html
  Web sites for handling recycled tubulin.
   http://www.bio.com/protocolstools/protocol.jhtml?id = p1426
  Protocol for tubulin/MAP co‐sedimentation under high ATP conditions and MAP/motor/tubulin co‐sedimentation.
   http://www.ciwemb.edu/labs/koshland/Protocols/MICROTUBULE/cyclingmicrotub.html
  In this protocol purified tubulin is stabilized with Taxol.
   http://www.bio.com/protocolstools/protocol.jhtml?id = p1434
  Web site for tubulin purification from 3 pig brains similar to .
   http://www.bio.com/protocolstools/protocol.jhtml?id = p1600
  Web page for microtubule protein preparation.
   http://www.bio.unc.edu/faculty/salmon/lab/protocolsporcinetubulin.html
   http://www.borisylab.northwestern.edu/pages/protocols.html
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