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Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity

Featured protocol,  Authors: Qiang Chen
Qiang ChenAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
Bio-protocol author page: a4118
Lingling Yu
Lingling YuAffiliation: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: 94116758@qq.com
Bio-protocol author page: a4119
Xiao Tan
Xiao Tan Affiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: xiao-tan@hotmail.com
Bio-protocol author page: a4120
 and Sen Liu
Sen LiuAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: senliu.ctgu@gmail.com
Bio-protocol author page: a4117
date: 2/20/2017, 39 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2140.

Brief version appeared in Sci Rep, Apr 2016
Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria are the simplest and one of the best studied models. They contain the three clock proteins KaiA, KaiB, and KaiC which can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol.

Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere

Featured protocol,  Authors: Carlos A. Ruiz-Pérez
Carlos A. Ruiz-PérezAffiliation 1: School of Biological Sciences, Georgia Institute of Technology, Ford Environmental Sciences & Technology Building, Atlanta, USA
Affiliation 2: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
For correspondence: cruizperez3@gatech.edu
Bio-protocol author page: a4126
 and María Mercedes Zambrano
María Mercedes ZambranoAffiliation: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
Bio-protocol author page: a4127
date: 2/20/2017, 30 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2142.

Brief version appeared in Appl Environ Microbiol, Jan 2016
The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for plant health. The characterization of microbial communities in diverse plants, such as Espeletia plants that are endemic to the Paramo ecosystem in the Andes Mountains, can shed light regarding possible interactions among microorganisms and microbial functional properties. Obtaining DNA from plant endophytic microbial communities involves various steps to ensure that samples are free of contamination from microorganisms present on the plant surface (epiphytes). Plant leaves are first surface sterilized, cut into pieces, homogenized using glass beads, and then used for DNA extraction using a commercially available kit. DNA samples are then quantified and analyzed using Qubit® 2.0 for use in PCR amplification of 16S rRNA genes.

Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies

Featured protocol,  Authors: Yung-Ning Chang
Yung-Ning ChangAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
Bio-protocol author page: a4040
Farooque R. Shaik
Farooque R. ShaikAffiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
Bio-protocol author page: a4041
Yvonne Neldner
Yvonne NeldnerAffiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4042
 and Eric R. Geertsma
Eric R. GeertsmaAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
For correspondence: geertsma@em.uni-frankfurt.de
Bio-protocol author page: a4043
date: 2/5/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2116.

Brief version appeared in Nat Struct Mol Biol, Oct 2015
The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, termed SLC26Dg, from Deinococcus geothermalis. Following these instructions we obtained well-folded and homogenous material of the membrane protein SLC26Dg and the nanobody Nb5776 that enabled us to crystallize the complex and determine its structure (Geertsma et al., 2015). The procedure may be adapted to purify and crystallize other membrane protein complexes.

Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract

Featured protocol,  Authors: Anna Waldhuber
Anna WaldhuberAffiliation: Molekulare Pädiatrie, Dr. von Haunersches Kinderspital, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4062
Manoj Puthia
Manoj PuthiaAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4063
Andreas Wieser
Andreas WieserAffiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4064
Catharina Svanborg
Catharina SvanborgAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4065
 and Thomas Miethke
Thomas Miethke Affiliation: Institute of Medical Microbiology and Hygiene, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany
For correspondence: thomas.miethke@medma.uni-heidelberg.de
Bio-protocol author page: a4056
date: 2/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2129.

Brief version appeared in J Clin Invest, Jul 2016
This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of both pattern recognition receptor systems (Cirl et al., 2008; Waldhuber et al., 2016). We infected wild type and knock-out mice with wildtype CFT073 and a mutant CFT073 strain lacking tcpC. This protocol describes how the mice were infected, how CFT073 was prepared and how the infection was monitored. The protocol was derived from our previously published work and allowed us to demonstrate that TcpC is a powerful virulence factor by increasing the bacterial burden of CFT073 in the urine and kidneys. Moreover, TcpC was responsible for the development of kidney abscesses since infection of mice with wildtype but not tcpC-deficient CFT073 mutants caused this complication.

Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers

Featured protocol,  Authors: Masaki Mizutani
Masaki MizutaniAffiliation: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
Bio-protocol author page: a4091
 and Makoto Miyata
Makoto MiyataAffiliation 1: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
Affiliation 2: The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka City University, Osaka, Japan
For correspondence: miyata@sci.osaka-cu.ac.jp
Bio-protocol author page: a711
date: 2/5/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2127.

Brief version appeared in mBio, Jun 2016
Dozens of Mycoplasma species, belonging to class Mollicutes form a protrusion at a pole as an organelle. They bind to solid surfaces through the organelle and glide in the direction by a unique mechanism including repeated cycles of bind, pull, and release with sialylated oligosaccharides on host animal cells. The mechanical characters are critical information to understand this unique mechanism involved in their infectious process. In this protocol, we describe a method to measure the force generated by Mycoplasma mobile, the fastest gliding species in Mycoplasma. This protocol should be useful for the studies of many kinds of gliding microorganisms.

Enriching Acidophilic Fe(II)-oxidizing Bacteria in No-flow, Fed-batch Systems

Featured protocol,  Authors: Yizhi Sheng
Yizhi ShengAffiliation 1: Department of Civil and Environmental Engineering, the Pennsylvania State University, PA, USA
Affiliation 2: School of Water Resources and Environment, China University of Geosciences, Beijing, China
For correspondence: shengyz@cugb.edu.cn
Bio-protocol author page: a4069
Bradley Kaley
Bradley KaleyAffiliation: Department of Civil and Environmental Engineering, the Pennsylvania State University, PA, USA
Bio-protocol author page: a4070
 and William D. Burgos
William D. BurgosAffiliation: Department of Civil and Environmental Engineering, the Pennsylvania State University, PA, USA
For correspondence: wdb3@engr.psu.edu
Bio-protocol author page: a4071
date: 2/5/2017, 83 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2130.

Brief version appeared in Appl Environ Microbiol, May 2016
Low-pH microbial Fe(II) oxidation occurs naturally in some Fe(II)-rich acid mine drainage (AMD) ecosystems across so-called terraced iron formations. Indigenous acidophilic Fe(II)-oxidizing bacterial communities can be incorporated into both passive and active treatments to remove Fe from the AMD solution. Here, we present a protocol of enriching acidophilic Fe(II)-oxidizing bacteria in no-flow, fed-batch systems. Mixed cultures of naturally occurring microbes are enriched from the fresh surface sediments at AMD sites using a chemo-static bioreactor (Eppendorf BioFlo®/Celligen® 115 Fermentor) with respect to constant stirring speed, temperature, pH and unlimited dissolved oxygen. Ferrous sulfate is discontinuously added to the reactor as the primary substrate to enrich for acidophilic Fe(II)-oxidizing bacteria. Successfully and efficiently enriching acidophilic Fe(II)-oxidizing bacteria helps to exploit this biogeochemical process into AMD treatment systems.

Determination of the in vitro Sporulation Frequency of Clostridium difficile

Featured protocol,  Authors: Adrianne N. Edwards
Adrianne N. EdwardsAffiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, GA, USA
For correspondence: anehrli@emory.edu
Bio-protocol author page: a4068
 and Shonna M. McBride
Shonna M. McBrideAffiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, GA, USA
Bio-protocol author page: a4067
date: 2/5/2017, 114 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2125.

Brief version appeared in Mol Microbiol, Jun 2016
The anaerobic, gastrointestinal pathogen, Clostridium difficile, persists within the environment and spreads from host-to-host via its infectious form, the spore. To effectively study spore formation, the physical differentiation of vegetative cells from spores is required to determine the proportion of spores within a population of C. difficile. This protocol describes a method to accurately enumerate both viable vegetative cells and spores separately and subsequently calculate a sporulation frequency of a mixed C. difficile population from various in vitro growth conditions (Edwards et al., 2016b).

Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity

Authors: Qiang Chen
Qiang ChenAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
Bio-protocol author page: a4118
Lingling Yu
Lingling YuAffiliation: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: 94116758@qq.com
Bio-protocol author page: a4119
Xiao Tan
Xiao Tan Affiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: xiao-tan@hotmail.com
Bio-protocol author page: a4120
 and Sen Liu
Sen LiuAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: senliu.ctgu@gmail.com
Bio-protocol author page: a4117
date: 2/20/2017, 39 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2140.

[Abstract] Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria ...

Endophytic Microbial Community DNA Extraction from the Plant Phyllosphere

Authors: Carlos A. Ruiz-Pérez
Carlos A. Ruiz-PérezAffiliation 1: School of Biological Sciences, Georgia Institute of Technology, Ford Environmental Sciences & Technology Building, Atlanta, USA
Affiliation 2: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
For correspondence: cruizperez3@gatech.edu
Bio-protocol author page: a4126
 and María Mercedes Zambrano
María Mercedes ZambranoAffiliation: Molecular Genetics, Corporación Corpogen, Bogotá, Colombia
Bio-protocol author page: a4127
date: 2/20/2017, 30 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2142.

[Abstract] The plant phyllosphere, which represents all plant parts that are above the ground, is considered one of the most extensive ecosystems to be colonized by microorganisms, both at the surface as epiphytes or as endophytes within the plant. These plant-associated microbial communities are reservoirs of microbial diversity and they can be important for ...

Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies

Authors: Yung-Ning Chang
Yung-Ning ChangAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
Bio-protocol author page: a4040
Farooque R. Shaik
Farooque R. ShaikAffiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
Bio-protocol author page: a4041
Yvonne Neldner
Yvonne NeldnerAffiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4042
 and Eric R. Geertsma
Eric R. GeertsmaAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
For correspondence: geertsma@em.uni-frankfurt.de
Bio-protocol author page: a4043
date: 2/5/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2116.

[Abstract] The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, ...

Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract

Authors: Anna Waldhuber
Anna WaldhuberAffiliation: Molekulare Pädiatrie, Dr. von Haunersches Kinderspital, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4062
Manoj Puthia
Manoj PuthiaAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4063
Andreas Wieser
Andreas WieserAffiliation: Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, München, Germany
Bio-protocol author page: a4064
Catharina Svanborg
Catharina SvanborgAffiliation: Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden
Bio-protocol author page: a4065
 and Thomas Miethke
Thomas Miethke Affiliation: Institute of Medical Microbiology and Hygiene, Medical Faculty of Mannheim, University of Heidelberg, Mannheim, Germany
For correspondence: thomas.miethke@medma.uni-heidelberg.de
Bio-protocol author page: a4056
date: 2/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2129.

[Abstract] This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of ...

Force Measurement on Mycoplasma mobile Gliding Using Optical Tweezers

Authors: Masaki Mizutani
Masaki MizutaniAffiliation: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
Bio-protocol author page: a4091
 and Makoto Miyata
Makoto MiyataAffiliation 1: Department of Biology, Graduate School of Science, Osaka City University, Osaka, Japan
Affiliation 2: The OCU Advanced Research Institute for Natural Science and Technology (OCARINA), Osaka City University, Osaka, Japan
For correspondence: miyata@sci.osaka-cu.ac.jp
Bio-protocol author page: a711
date: 2/5/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2127.

[Abstract] Dozens of Mycoplasma species, belonging to class Mollicutes form a protrusion at a pole as an organelle. They bind to solid surfaces through the organelle and glide in the direction by a unique mechanism including repeated cycles of bind, pull, and release with sialylated oligosaccharides on host animal cells. The mechanical characters are critical ...

Enriching Acidophilic Fe(II)-oxidizing Bacteria in No-flow, Fed-batch Systems

Authors: Yizhi Sheng
Yizhi ShengAffiliation 1: Department of Civil and Environmental Engineering, the Pennsylvania State University, PA, USA
Affiliation 2: School of Water Resources and Environment, China University of Geosciences, Beijing, China
For correspondence: shengyz@cugb.edu.cn
Bio-protocol author page: a4069
Bradley Kaley
Bradley KaleyAffiliation: Department of Civil and Environmental Engineering, the Pennsylvania State University, PA, USA
Bio-protocol author page: a4070
 and William D. Burgos
William D. BurgosAffiliation: Department of Civil and Environmental Engineering, the Pennsylvania State University, PA, USA
For correspondence: wdb3@engr.psu.edu
Bio-protocol author page: a4071
date: 2/5/2017, 83 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2130.

[Abstract] Low-pH microbial Fe(II) oxidation occurs naturally in some Fe(II)-rich acid mine drainage (AMD) ecosystems across so-called terraced iron formations. Indigenous acidophilic Fe(II)-oxidizing bacterial communities can be incorporated into both passive and active treatments to remove Fe from the AMD solution. Here, we present a protocol of enriching acidophilic ...

Determination of the in vitro Sporulation Frequency of Clostridium difficile

Authors: Adrianne N. Edwards
Adrianne N. EdwardsAffiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, GA, USA
For correspondence: anehrli@emory.edu
Bio-protocol author page: a4068
 and Shonna M. McBride
Shonna M. McBrideAffiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Emory Antibiotic Resistance Center, Atlanta, GA, USA
Bio-protocol author page: a4067
date: 2/5/2017, 114 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2125.

[Abstract] The anaerobic, gastrointestinal pathogen, Clostridium difficile, persists within the environment and spreads from host-to-host via its infectious form, the spore. To effectively study spore formation, the physical differentiation of vegetative cells from spores is required to determine the proportion of spores within a population of C. difficile. This ...

Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor

Authors: Stephanie Beltz
Stephanie BeltzAffiliation: Pharmazeutisches Institut der Universität Tübingen, Pharmazeutische Biochemie, Tübingen, Germany
For correspondence: stephanie.beltz@uni-tuebingen.de
Bio-protocol author page: a4015
 and Joachim E. Schultz
Joachim E. SchultzAffiliation: Pharmazeutisches Institut der Universität Tübingen, Pharmazeutische Biochemie, Tübingen, Germany
For correspondence: joachim.schultz@uni-tuebingen.de
Bio-protocol author page: a4016
date: 1/20/2017, 205 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2112.

[Abstract] The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins....

Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry

Authors: Manish Bhuwan*
Manish BhuwanAffiliation: Molecular Infection and Functional Biology Laboratory, Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India
Bio-protocol author page: a4003
Madhuri Suragani*$
Madhuri SuraganiAffiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad, Professor C.R. Rao Road, Hyderabad, India
Bio-protocol author page: a4004
Nasreen Z. Ehtesham
Nasreen Z. EhteshamAffiliation: Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India
For correspondence: nzehtesham@gmail.com
Bio-protocol author page: a4005
 and Seyed E. Hasnain
Seyed E. HasnainAffiliation 1: Molecular Infection and Functional Biology Laboratory, Kusuma School of Biological Sciences, Indian Institute of Technology, New Delhi, India
Affiliation 2: Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Professor C.R. Rao Road, Hyderabad, India
Affiliation 3: JH Institute of Molecular Medicine, Jamia Hamdard, Hamdard Nagar, New Delhi, India
For correspondence: seyedhasnain@gmail.com
Bio-protocol author page: a4006
 (*contributed equally to this work, $Deceased) date: 1/20/2017, 213 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2107.

[Abstract] The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure ...

Quantification of Triphenyl-2H-tetrazoliumchloride Reduction Activity in Bacterial Cells

Authors: Roberto Defez
Roberto DefezAffiliation: Institute of Biosciences and BioResources, Via P. Castellino 111, Naples, Italy
Bio-protocol author page: a4052
Anna Andreozzi
Anna AndreozziAffiliation: Institute of Biosciences and BioResources, Via P. Castellino 111, Naples, Italy
Bio-protocol author page: a4053
 and Carmen Bianco
Carmen BiancoAffiliation: Institute of Biosciences and BioResources, Via P. Castellino 111, Naples, Italy
For correspondence: carmen.bianco@ibbr.cnr.it
Bio-protocol author page: a4054
date: 1/20/2017, 186 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2115.

[Abstract] This protocol describes the use of the 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) salt to evaluate the cell redox potential of rhizobia cells. The production of brightly colored and insoluble 1,3,5-Triphenyltetrazolium formazan arising from TTC reduction is irreversible and can be easily quantified using a spectrophotometer. This protocol allows ...
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[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fanglian09@gmail.com
Bio-protocol author page: a9
date: 2/5/2011, 82765 views, 31 Q&A
DOI: https://doi.org/10.21769/BioProtoc.30.

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits....

[Bio101] E. coli Genomic DNA Extraction Updates
The author made some updates (highlighted in blue) to the protocol on 09/12/2016.

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a9
date: 7/20/2011, 75542 views, 46 Q&A
DOI: https://doi.org/10.21769/BioProtoc.97.

[Abstract] This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits....

[Bio101] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 22389 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.39.

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid....

In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
date: 6/5/2012, 21038 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

[Bio101] Making Yeast Competent Cells and Yeast Cell Transformation

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/20/2011, 20369 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.96.

[Abstract] This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells....

Spot Assay for Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2012, 17549 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....

[Bio101] Purification of Adenovirus by Cesium Chloride Density Gradients

Author: Huan Pang
Huan PangAffiliation: Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, USA
For correspondence: pang_huan@hotmail.com
Bio-protocol author page: a48
date: 4/5/2012, 17138 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.201.

[Abstract] Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography ...

Culture and Detection of Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (BCG)

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 1/20/2012, 16439 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.49.

[Abstract] Mycobacterium tuberculosis (MTB) is the bacterial pathogen responsible for tuberculosis, a human pulmonary infectious disease. Mycobacterium bovis (BCG) is the causative agent of tuberculosis in cattle, and is often used as the vaccine stain in humans. Specific recipes and methods for culture of MTB ...

[Bio101] Yeast Vacuole Staining with FM4-64

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 14022 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.18.

[Abstract] The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, ...

[Bio101] Purification of 6x His-tagged Protein (from E. coli)

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 12892 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.8.

[Abstract] A polyhistidine-tag is an amino acid motif that contains at least six histidine (His) residues, usually at the N- or C-terminus of the protein. This tag can also be referred to as a hexa histidine-tag or a 6x His-tag. The protocol described here has been developed to purify His-tagged proteins from ...
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