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Phenotypes observed for certain Moraxella catarrhalis wild-type strains or mutants may be caused by a variety of factors including alteration of outer membrane protein composition. Examination of the outer membrane protein profiles may be a valuable tool to identify changes in outer membrane compositions of these strains. Here we describe a method to isolate and analyse M. catarrhalis fractions highly enriched for membrane proteins.

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Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles

Microbiology > Microbial proteomics > Membrane proteins
Authors: Stefan P.W. de Vries
Stefan P.W. de VriesAffiliation: Laboratory of Pediatric Infectious Diseases, Radboud University Medical Centre, Nijmegen, The Netherlands
Bio-protocol author page: a949
 and Hester J. Bootsma
Hester J. BootsmaAffiliation: Laboratory of Pediatric Infectious Diseases, Radboud University Medical Centre, Nijmegen, The Netherlands
For correspondence: hester.bootsma@radboudumc.nl
Bio-protocol author page: a950
Vol 3, Iss 21, 11/5/2013, 2539 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.957

[Abstract] Phenotypes observed for certain Moraxella catarrhalis wild-type strains or mutants may be caused by a variety of factors including alteration of outer membrane protein composition. Examination of the outer membrane protein profiles may be a valuable tool to identify changes in outer membrane compositions of these strains. Here we describe a method to isolate and analyse M. catarrhalis fractions highly enriched for membrane proteins.

Keywords: Moraxella catarrhalis, Outer membrane vesicles, Pathogenesis, Virulence

Materials and Reagents

  1. Brain heart infusion (BHI) (Becton Dickinson and Company, catalog number: 237500) broth and BHI agar plates
  2. Antibiotics
    e.g. spectinomycin or kanamycin (Merck KgaA, Calbiochem, catalog numbers: 567570-10 and 420311-5)
  3. PBS
  4. Bovine skin gelatin (Sigma-Aldrich, catalog number: G9382-100G)
  5. ReadyPrep Protein Extraction Kit (membrane I) (Bio-Rad, catalog number: 1632088)
  6. Glass beads, acid-washed (150-212 μm) (Sigma-Aldrich, catalog number: G1145)
  7. 2D-Quant Kit (General Electric Company, catalog number: 80-6483-56)
  8. Mini-protean TGX precast gels, 4-15% (Bio-Rad, catalog number: 456-1083)
  9. Colloidal Coomassie staining (Pink et al., 2012)

Equipment

  1. CO2 incubator
  2. Benchtop Incubator Shaker
  3. Centrifuge
  4. TissueLyser LT (QIAGEN, model: 85600)

Procedure

  1. M. catarrhalis strains were inoculated on brain heart infusion (BHI) plates (supplemented with antibiotics when appropriate), and grown overnight at 37 °C in an atmosphere containing 5% CO2.
  2. Bacteria were harvested from plate and resuspended in PBS supplemented with 0.15% gelatin (PBS-G). This suspension was used to inoculate BHI broth to an OD620 nm of ~ 0.05 and grown at 37 °C at 200-250 rpm until OD620 nm of 1.0 to 1.2 (mid-log). Use of different growth media is possible, but may affect outer membrane protein profiles. Always use the same growth media when comparing outer membrane protein profiles of different strains.
  3. Subsequently, bacteria were harvested by centrifugation for 10 minutes at 3,200 x g.
  4. Outer membranes were isolated using the ReadyPrep Membrane I kit according to manufacturer’s instructions.
  5. During the lysis procedure, 50 mg acid-washed glass beads (150-212 μm) were added for homogenization with the TissueLyser LT. The TissueLyser was operated 5 times for 1 minute at 50 Hz.
  6. Suspensions were chilled on ice for 1 minute between every TissueLyser step.
  7. Protein quantification was performed using the 2D-Quant Kit according to manufacturer’s instructions.
  8. Five microgram of outer membrane preparations were separated on a 4-15% TGX gel and analyzed by colloidal Coomassie staining (Pink et al., 2010).

Acknowledgments

This protocol was published in: de Vries et al. (2013). This study was financially supported by Vienna Spot of Excellence (VSOE) grant (ID337956).

References

  1. de Vries, S. P., Burghout, P., Langereis, J. D., Zomer, A., Hermans, P. W. and Bootsma, H. J. (2013). Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions. Mol Microbiol 87(1): 14-29.    
  2. Pink, M., Verma, N., Rettenmeier, A. W. and Schmitz-Spanke, S. (2010). CBB staining protocol with higher sensitivity and mass spectrometric compatibility. Electrophoresis 31(4): 593-598.   


How to cite: Vries, S. P. and Bootsma, H. J. (2013). Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles. Bio-protocol 3(21): e957. DOI: 10.21769/BioProtoc.957; Full Text



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