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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.

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.

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, ...

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 ...

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 ...

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 ...

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 ...

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....

In vitro Dephosphorylation Assay of c-Myc

Authors: Peng Liao
Peng LiaoAffiliation: Department of Central Laboratory, Tongji University, Shanghai, China
For correspondence: liaopengchina@gmail.com
Bio-protocol author page: a3718
Weichao Wang
Weichao WangAffiliation: Department of Central Laboratory, Tongji University, Shanghai, China
Bio-protocol author page: a3719
 and Xin Ge
Xin GeAffiliation: Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital of Tongji University, Shanghai, China
Bio-protocol author page: a3720
date: 1/20/2017, 217 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2011.

[Abstract] This protocol describes experimental procedures for in vitro dephosphorylation assay of human protein c-Myc. This protocol can be adapted to detect phosphatase activity of other Ser/Thr phosphatases....

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 ...

Bioassay of Xanthomonas albilineans Attachment on Sugarcane Leaves

Authors: Imène Mensi
Imène MensiAffiliation: Department of Biological Sciences and Plant Protection, Higher Agronomic Institute of Chott-Mariem, Sousse, Tunisia
Bio-protocol author page: a3989
Jean-Heinrich Daugrois
Jean-Heinrich DaugroisAffiliation: UMR BGPI, CIRAD, Montpellier, France
Bio-protocol author page: a3990
 and Philippe Rott
Philippe RottAffiliation: Department of Plant Pathology, University of Florida, Everglades Research and Education Center, Belle Glade, USA
For correspondence: pcrott@ufl.edu
Bio-protocol author page: a3991
date: 1/20/2017, 200 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2111.

[Abstract] Sugarcane (interspecific hybrids of Saccharum species) is an economically important crop that provides 70% of raw table sugar production worldwide and contributes, in some countries, to bioethanol and electricity production. Leaf scald, caused by the bacterial plant pathogen Xanthomonas albilineans, ...

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 ...
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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] GST-Pull Down Protocol

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2012, 39576 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.177.

[Abstract] GST-Pull down assay is an effective way to examine the direct binding of two proteins in vitro. This protocol is based on GST pull down system from GE healthcare, and uses the binding of unplugged/MuSK receptor and Wnt ligand as an example to illustrate the detailed procedure....

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] 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 ...

[Bio101] RbCl Super Competent Cells

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 6/5/2011, 10748 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.76.

[Abstract] This method is used to inexpensively prepare home-made competent cells of E. coli. The transformation efficiency is at the high end of chemical-efficient competent cells, and close to library-efficient competent cells....

[Bio101] Expression and Purification of GST-tagged Proteins from E. coli

Author: Lin Fang
Lin FangAffiliation: Department of Pediatrics, School of Medicine, Stanford University, Stanford, USA
For correspondence: cheerfulfang@hotmail.com
Bio-protocol author page: a20
date: 9/20/2011, 10441 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.132.

[Abstract] This protocol describes a method for the small and large-scale expression and purification of GST proteins. Due to the diverse nature of proteins, a small-scale expression and purification test is always recommended....

[Bio101] The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells

Author: Hogune Im date: 10/20/2011, 9870 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.143.

[Abstract] This protocol differs from other procedures in that the bacterial culture is grown at 18 °C rather than the conventional 37 °C. Otherwise, the protocol is unremarkable and follows a fairly standard course. Why growing the cells at low temperature should affect the efficiency of transformation is unknown. ...

KMnO4 Footprinting

Authors: Ümit Pul
Ümit PulAffiliation: Molecular Biology of Bacteria, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
Bio-protocol author page: a137
Reinhild Wurm
Reinhild WurmAffiliation: Molecular Biology of Bacteria, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
Bio-protocol author page: a138
 and Rolf Wagner
Rolf WagnerAffiliation: Molecular Biology of Bacteria, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
For correspondence: r.wagner@rz.uni-duesseldorf.de
Bio-protocol author page: a139
date: 11/5/2012, 8352 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.280.

[Abstract] The KMnO4 footprinting method offers a rapid and easy way to detect and localize single-stranded regions within a duplex DNA molecule, such as it occurs for instance within an actively transcribing RNA polymerase-DNA complex or during R-loop formation in DNA-RNA hybrid structures. The method is based ...

Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins

Authors: Timo A. Lehti
Timo A. LehtiAffiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland
For correspondence: timo.lehti@helsinki.fi
Bio-protocol author page: a809
 and Benita Westerlund-Wikström
Benita Westerlund-WikströmAffiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a810
date: 9/5/2013, 7760 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.888.

[Abstract] This simple protocol describes how to detect antigens from agar-grown bacterial colonies transferred to nitrocellulose using specific antibodies. The protocol is well suitable for detection of bacterial proteins exposed on the cell surface or secreted to the extracellular space and it can be modified ...
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