Analyses of Mycobacterium tuberculosis Proteins

Michelle H. Larsen1, Karolin Biermann1, William R. Jacobs,1

1 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10A.4
DOI:  10.1002/9780471729259.mc10a04s6
Online Posting Date:  August, 2007
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Abstract

This unit includes protocols for the isolation of proteins from Mycobacterium tuberculosis. Considerations for working with M. tuberculosis at Biosafety Level 3 containment are also discussed. Curr. Protoc. Microbiol. 6:10A.4.1‐10A.4.5. © 2007 by John Wiley & Sons, Inc.

Keywords: tuberculosis; TB; protein; culture filtrate; Sauton

     
 
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Table of Contents

  • Introduction
  • Safety Considerations
  • Basic Protocol 1: Extraction of M. tuberculosis Proteins Using the Glass Bead Method
  • Basic Protocol 2: Preparation of Culture Filtrates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Extraction of M. tuberculosis Proteins Using the Glass Bead Method

  Materials
  • Mycobacteria (unit 10.1)
  • Phosphate buffered saline (PBS; appendix 2A)
  • Protein extraction buffer (see recipe), ice cold
  • 2× SDS sample buffer ( appendix 2A)
  • 15‐ml screw‐cap conical tubes
  • Refrigerated tabletop centrifuge
  • Vortex and tube adaptor
  • 2‐ml screw‐cap tubes (Sarstedt)
  • Microcentrifuge
  • Glass beads (106‐µm, Sigma cat. no. G4649)
  • 95°C water bath

Basic Protocol 2: Preparation of Culture Filtrates

  Materials
  • Bacterial culture in Sauton medium (UNIT 10A.1)
  • 50‐ml conical tubes
  • Sterile cell scraper
  • Refrigerated tabletop centrifuge
  • Filter units (0.45‐ and 0.2‐µm)
  • Cell concentrator (Amicon Ultrafiltration unit or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
   Schwebach, J.R., Jacobs, W.R. Jr., and Casadevall, A. 2001. Sterilization of Mycobacterium tuberculosis Erdman samples by antimicrobial fixation in a Biosafety Level 3 laboratory. J. Clin. Microbiol. 39:769‐771.
   Braunstein, M., Espinosa, B.J., Chan, J., Belisle, J.T., and Jacobs, W.R. Jr. 2003. SecA2 functions in the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis. Mol. Microbiol. 48:453‐464.
   Kriakov, J., Lee, S., and Jacobs, W.R. Jr. 2003. Identification of a regulated alkaline phosphatase, a cell surface–associated lipoprotein, in Mycobacterium smegmatis. J. Bacteriol. 185:4983‐4991.
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