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Volume:3  Issue:21

November 05, 2013

Biochemistry

Quantification of Bacterial Fatty Acids by Extraction and Methylation

Authors: Mark Politz
Mark PolitzAffiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA
Bio-protocol author page: a938
Rebecca Lennen
Rebecca LennenAffiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA
Bio-protocol author page: a939
 and Brian Pfleger
Brian PflegerAffiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA
For correspondence: pfleger@engr.wisc.edu
Bio-protocol author page: a557
date: 11/5/2013, 5537 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.950 

[Abstract] This protocol describes two similar methods for the extraction and methylation of fatty acids from bacterial cultures. The acid derivatization protocol (Lennen et al., 2013; Bligh and Dyer, 1959) results in the extraction and methylation of all fatty ...

Article identifier: bio-protocol.org/e950

β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)

Author: Ralph T. Böttcher
Ralph T. BöttcherAffiliation: Molecular Medicine Division, Max Planck Institute for Biochemistry, Martinsried, Germany
For correspondence: rboettch@biochem.mpg.de
Bio-protocol author page: a962
date: 11/5/2013, 3803 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.962 

[Abstract] Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions ...

Article identifier: bio-protocol.org/e962

Cell Biology

Ki67 Immunofluorescence on Bovine Cell Lines

Authors: Justine Marsolier
Justine MarsolierAffiliation: Epigenetics and Cell Fate, UMR 7216 CNRS, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
Bio-protocol author page: a945
Souhila Medjkane
Souhila MedjkaneAffiliation: Epigenetics and Cell Fate, UMR 7216 CNRS, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
Bio-protocol author page: a946
Martine Perichon
Martine PerichonAffiliation: Epigenetics and Cell Fate, UMR 7216 CNRS, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
Bio-protocol author page: a947
 and Jonathan B. Weitzman
Jonathan B. WeitzmanAffiliation: Epigenetics and Cell Fate, UMR 7216 CNRS, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
For correspondence: jonathan.weitzman@univ-paris-diderot.fr
Bio-protocol author page: a948
date: 11/5/2013, 2794 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.958 

[Abstract] This is a rapid protocol to test the effects of drugs treatment on bovine cell replication using Ki67 staining. Ki67 is associated with cell proliferation and is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent ...

Article identifier: bio-protocol.org/e958

Immunology

Bacterial Counts in Spleen

Authors: Elías Barquero-Calvo
Elías Barquero-CalvoAffiliation: Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica
Bio-protocol author page: a954
Carlos Chacón-Díaz
Carlos Chacón-DíazAffiliation: Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
Bio-protocol author page: a958
Esteban Chaves-Olarte
Esteban Chaves-OlarteAffiliation: Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
Bio-protocol author page: a960
 and Edgardo Moreno
Edgardo MorenoAffiliation: Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica
For correspondence: emoreno@racsa.co.cr
Bio-protocol author page: a961
date: 11/5/2013, 8029 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.954 

[Abstract] Bacterial loads can be determined as colony forming units (CFU) at any point of the infection by culturing spleen homogenates on agar plates. This is a reliable method for comparing the kinetics of infection in various mouse strains, estimating the virulence ...

Article identifier: bio-protocol.org/e954

Microbiology

H2O2 Kill Assays of Biofilm Bacteria

Authors: Malika Khakimova
Malika KhakimovaAffiliation: Department of Microbiology, McGill University, Montreal, Canada
Bio-protocol author page: a951
 and Dao Nguyen
Dao NguyenAffiliation: Department of Medicine, McGill University, Montreal, Canada
For correspondence: dao.nguyen@mcgill.ca
Bio-protocol author page: a952
date: 11/5/2013, 3537 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.952 

[Abstract] Ubiquitous in nature and often surface associated, biofilms cause numerous chronic human infections. Biofilms are structured multicellular bacterial communities where cells are entrapped in a polymer matrix. Bacteria growing as biofilms are characterized ...

Article identifier: bio-protocol.org/e952

H2O2 Kill Assays of Planktonic Stationary Phase Bacteria

Authors: Malika Khakimova
Malika KhakimovaAffiliation: Department of Microbiology, McGill University, Montreal, Canada
Bio-protocol author page: a951
 and Dao Nguyen
Dao NguyenAffiliation: Department of Medicine, McGill University, Montreal, Canada
For correspondence: dao.nguyen@mcgill.ca
Bio-protocol author page: a952
date: 11/5/2013, 3553 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.953 

[Abstract] Stationary phase bacteria are highly tolerant to hydrogen peroxide. This protocol was developed to test the susceptibility to hydrogen peroxide killing in different Pseudomonas aeruginosa strains. This assay provides a reliable way to measure killing ...

Article identifier: bio-protocol.org/e953

Adhesion of Moraxella catarrhalis to Respiratory Tract Epithelial Cells

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
date: 11/5/2013, 3700 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.956 

[Abstract] Moraxella catarrhalis is a human-restricted pathogen that is responsible for respiratory tract infections such as childhood otitis media (OM) and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Successful colonization and infection ...

Article identifier: bio-protocol.org/e956

Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles

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
date: 11/5/2013, 3184 views, 0 Q&A
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 ...

Article identifier: bio-protocol.org/e957

Immunoplaque Assay (Influenza Virus)

Authors: Longping V. Tse
Longping V. TseAffiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, USA
For correspondence: lt273@cornell.edu
Bio-protocol author page: a940
Yueting Zhang
Yueting ZhangAffiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, USA
Bio-protocol author page: a941
 and Gary R. Whittaker
Gary R. WhittakerAffiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, USA
For correspondence: grw7@cornell.edu
Bio-protocol author page: a942
date: 11/5/2013, 7217 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.959 

[Abstract] Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay ...

Article identifier: bio-protocol.org/e959

Neuroscience

Dissection of Different Areas from Mouse Hippocampus

Author: Faraz A. Sultan
Faraz A. SultanAffiliation: Neurobiology Department, University of Alabama at Birmingham, Birmingham, USA
For correspondence: faraz@uab.edu
Bio-protocol author page: a953
date: 11/5/2013, 6581 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.955 

[Abstract] The hippocampus modulates a number of modules including memory consolidation, spatial navigation, temporal processing and emotion. A banana-shaped structure, the hippocampus is constituted of morphologically distinct subregions including the dentate ...

Article identifier: bio-protocol.org/e955

Mitochondrial Isolation and Purification from Mouse Spinal Cord

Authors: Philippe A. Parone
Philippe A. ParoneAffiliation: Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego (UCSD), La Jolla, CA, USA
For correspondence: philippeparone@gmail.com
Bio-protocol author page: a933
Sandrine Da Cruz
Sandrine Da CruzAffiliation: Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego (UCSD), La Jolla, CA, USA
Bio-protocol author page: a934
 and Don W. Cleveland
Don W. ClevelandAffiliation: Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego (UCSD), La Jolla, CA, USA
Bio-protocol author page: a935
date: 11/5/2013, 3604 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.961 

[Abstract] Mitochondria are eukaryotic organelles that play a crucial role in several cellular processes, including energy production, β-oxidation of fatty acids and regulation of calcium homeostasis. In the last 20 years there has been a hightened interest in ...

Article identifier: bio-protocol.org/e961

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Implantation of Dkk-1-soaked Beads into the Neural Tube of Chicken Embryos

Authors: Almudena Martinez-Ferre
Almudena Martinez-FerreAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
Bio-protocol author page: a967
Maria Navarro-Garberí
Maria Navarro-GarberíAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
Bio-protocol author page: a968
Carlos Bueno
Carlos BuenoAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
Bio-protocol author page: a969
 and Salvador Martinez
Salvador MartinezAffiliation: Institute of Neurosciences, Miguel Hernández University-Spanish National Research Council, San Juan, Alicante, Spain
For correspondence: smartinez@umh.es
Bio-protocol author page: a970
date: 11/5/2013, 4607 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.963 

[Abstract] Chick embryos are known to be a powerful system to test gene function due to the “in vivo” accessibility, short time for results retrieval and the possibility to perform a large number of experiments. Synthetic micro-beads soaked in morphogenetic signals ...

Article identifier: bio-protocol.org/e963

NMDA-induced Excitotoxicity and Lactate Dehydrogenase Assay in Primary Cultured Neurons

Authors: Shu Zhang
Shu ZhangAffiliation: Brain Research Centre, University of British Columbia, Vancouver, Canada
Bio-protocol author page: a963
 and Yu Tian Wang
Yu Tian WangAffiliation: Brain Research Centre, University of British Columbia, Vancouver, Canada
For correspondence: ytwang@brain.ubc.ca
Bio-protocol author page: a964
date: 11/5/2013, 4750 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.965 

[Abstract] N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitotoxicity is thought to contribute to the pathogenesis of a large number of chronic neurodegenerative disorders (such as Alzheimer’s and Huntington’s diseases to mental illnesses) in addition to ...

Article identifier: bio-protocol.org/e965

Plant Science

Ovule Clearing Method for Solanaceous Species

Authors: Audrey Loubert-Hudon
Audrey Loubert-HudonAffiliation: Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Montréal, Québec, Canada
Bio-protocol author page: a943
 and Daniel P. Matton
Daniel P. MattonAffiliation: Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, Montréal, Québec, Canada
For correspondence: dp.matton@umontreal.ca
Bio-protocol author page: a944
date: 11/5/2013, 3129 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.951 

[Abstract] Depending on the species, embryo sacs can be difficult to observe. Ovule clearing allows precise observation of whole tissues under Differential interference contrast (DIC) microscopy. The use of Methyl Salicylate as a clearing agent has proved to be ...

Article identifier: bio-protocol.org/e951

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Visualization and Quantification of Actin Dynamics in Rice Protoplasts

Authors: Yurong Xie
Yurong XieAffiliation: Key laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a965
 and Shanjin Huang
Shanjin HuangAffiliation: Key laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
For correspondence: sjhuang@ibcas.ac.cn
Bio-protocol author page: a966
date: 11/5/2013, 2954 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.964 

[Abstract] Direct visualization of the organization and dynamics of the actin cytoskeleton in rice cells is essential to understand its roles in regulating rice growth and development. Visualization of actin dynamics in protoplasts transformed with actin probe ...

Article identifier: bio-protocol.org/e964

Stem Cell

In vivo BrdU Incorporation Assay for Murine Hematopioetic Stem Cells

Authors: Ningfei An
Ningfei AnAffiliation: Division of Hematology-Oncology, Department of Medicine, Medical University of South Carolina, Charleston, USA
Bio-protocol author page: a936
 and Yubin Kang
Yubin KangAffiliation: Division of Hematology-Oncology, Department of Medicine, Medical University of South Carolina, Charleston, USA
For correspondence: kangy@musc.edu
Bio-protocol author page: a937
date: 11/5/2013, 5100 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.960 

[Abstract] Bromodeoxyuridine (BrdU) is a thymidine analog that is incorporated into DNA during the S-phase of the cell cycle. As such, BrdU incorporation can be used to quantify the number of cells that are in S-phase in the time period during which BrdU is available. ...

Article identifier: bio-protocol.org/e960