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Biochemistry

Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth

Authors: Natalia V. Andreeva
Natalia V. AndreevaAffiliation: Laboratory of Stem and Progenitor Cell Biology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Bio-protocol author page: a4766
 and Alexander V. Belyavsky
Alexander V. BelyavskyAffiliation: Laboratory of Stem and Progenitor Cell Biology, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
For correspondence: abelyavs@yahoo.com
Bio-protocol author page: a4767
,  date: 7/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2374.

[Abstract] Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained by the loss immobilized proteins due to their ...

Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor

Authors: Rajan Lamichhane
Rajan LamichhaneAffiliation: Department of Integrative Structural & Computational Biology, the Scripps Research Institute, La Jolla, CA, USA
Bio-protocol author page: a4660
, Jeffrey J. Liu
Jeffrey J. LiuAffiliation 1: Department of Integrative Structural & Computational Biology, the Scripps Research Institute, La Jolla, CA, USA
Affiliation 2: Max Planck Institute for Biochemistry, Martinsried, Germany
Bio-protocol author page: a4661
, Raymond F. Pauszek III
Raymond F. Pauszek IIIAffiliation: Department of Integrative Structural & Computational Biology, the Scripps Research Institute, La Jolla, CA, USA
Bio-protocol author page: a4662
 and David P. Millar
David P. MillarAffiliation: Department of Integrative Structural & Computational Biology, the Scripps Research Institute, La Jolla, CA, USA
For correspondence: millar@scripps.edu
Bio-protocol author page: a4663
,  date: 6/20/2017, 249 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2332.

[Abstract] Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence ...

Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein

Authors: Byron Carpenter
Byron CarpenterAffiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, UK
Present address: Warwick Integrative Synthetic Biology Centre, School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry, UK
For correspondence: b.carpenter@warwick.ac.uk
Bio-protocol author page: a4379
 and Christopher G. Tate
Christopher G. TateAffiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, UK
For correspondence: cgt@mrc-lmb.cam.ac.uk
Bio-protocol author page: a4380
,  date: 4/20/2017, 903 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2234.

[Abstract] G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR–G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are challenging ...

Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila for Negative Stain Electron Microscopy Studies

Authors: Tomoko Kubori
Tomoko KuboriAffiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Japan
For correspondence: tkubori@biken.osaka-u.ac.jp
Bio-protocol author page: a4367
 and Hiroki Nagai
Hiroki NagaiAffiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Japan
Bio-protocol author page: a4368
,  date: 4/20/2017, 434 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2229.

[Abstract] Legionella possesses a pivotal secretion machinery to deliver virulence factors to eukaryotic host cells. In this protocol, we describe the procedure for isolation of the native core complex of the Dot/Icm type IV secretion system from L. pneumophila aiming to perform biochemical and transmission electron microscopy analyses....

Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography

Authors: Valentina La Verde
Valentina La VerdeAffiliation: Department of Biotechnology, University of Verona, Verona, Italy
Bio-protocol author page: a4369
, Paola Dominici
Paola DominiciAffiliation: Department of Biotechnology, University of Verona, Verona, Italy
Bio-protocol author page: a4370
 and Alessandra Astegno
Alessandra AstegnoAffiliation: Department of Biotechnology, University of Verona, Verona, Italy
For correspondence: alessandra.astegno@univr.it
Bio-protocol author page: a4371
,  date: 4/20/2017, 694 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2230.

[Abstract] Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties (i.e., frictional ...

Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies

Authors: Shengping Li
Shengping LiAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4023
, Yanhe Zhao
Yanhe ZhaoAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4024
 and Yunkun Wu
Yunkun WuAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
For correspondence: wuyk@fjirsm.ac.cn
Bio-protocol author page: a4025
,  date: 2/5/2017, 833 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2122.

[Abstract] GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is about ...

PRODIGY: A Contact-based Predictor of Binding Affinity in Protein-protein Complexes

Authors: Anna Vangone
Anna VangoneAffiliation: Computational Structural Biology group, Bijvoet Center for Biomolecular Research, Faculty of Science Chemistry, Utrecht University, Utrecht, the Netherlands
For correspondence: a.vangone@uu.nl
Bio-protocol author page: a4057
 and Alexandre M. J. J. Bonvin
Alexandre M. J. J. BonvinAffiliation: Computational Structural Biology group, Bijvoet Center for Biomolecular Research, Faculty of Science Chemistry, Utrecht University, Utrecht, the Netherlands
For correspondence: a.m.j.j.bonvin@uu.nl
Bio-protocol author page: a4058
,  date: 2/5/2017, 1015 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2124.

[Abstract] Biomolecular interactions between proteins regulate and control almost every biological process in the cell. Understanding these interactions is therefore a crucial step in the investigation of biological systems and in drug design. Many efforts have been devoted to unravel principles of protein-protein interactions. Recently, we introduced a simple ...

Protocol for Molecular Dynamics Simulations of Proteins

Authors: MNV Prasad Gajula
MNV Prasad GajulaAffiliation 1: Institute of Biotechnology, PJTSAU, Rajendra Nagar, Hyderabad, India
Affiliation 2: Bioclues.org, Kukatpally, Telangana, India
For correspondence: gajula.ibt@gmail.com
Bio-protocol author page: a3836
, Anuj Kumar
Anuj KumarAffiliation: Bioinformatics center, Uttarakhand Council for Biotechnology,, Dehradun, India
Bio-protocol author page: a3837
 and Johny Ijaq
Johny IjaqAffiliation: Department of Zoology, Osmania University, Hyderabad, India
Bio-protocol author page: a3838
,  date: 12/5/2016, 2163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2051.

[Abstract] Molecular dynamics (MD) simulations have become one of the most important tools in understanding the behavior of bio-molecules on nanosecond to microsecond time scales. In this protocol, we provide a general approach and standard setup protocol for MD simulations by using the Gromacs MD suite. ...

Heterologous Expression and Purification of Catalytic Domain of CESA1 from Arabidopsis thaliana

Authors: Venu Gopal Vandavasi
Venu Gopal VandavasiAffiliation: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA
Bio-protocol author page: a3603
 and Hugh O’ Neill
Hugh O’ NeillAffiliation: Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA
For correspondence: oneillhm@ornl.gov
Bio-protocol author page: a3604
,  date: 10/20/2016, 1079 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1965.

[Abstract] Heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant ...

Trypsin Sensitivity Assay to Study the Folding Status of Proteins

Authors: Satoshi Ninagawa
Satoshi NinagawaAffiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan
Bio-protocol author page: a3569
 and Kazutoshi Mori
Kazutoshi MoriAffiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan
For correspondence: mori@upr.biophys.kyoto-u.ac.jp
Bio-protocol author page: a3570
,  date: 10/5/2016, 1007 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1953.

[Abstract] This protocol aims to evaluate folding status of proteins, utilizing trypsin sensitivity. Unfolded/misfolded proteins are more susceptible to trypsin than folded proteins, because trypsin easily accesses and cleaves loosely folded parts of proteins. This method is especially useful to compare tightness of the folding among wild-type and mutant proteins. ...
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Analysis of L- and D-Amino Acids Using UPLC

Authors: Yuta Mutaguchi
Yuta MutaguchiAffiliation: Department of Biotechnology, Akita Prefectural University, Akita City, Japan
Bio-protocol author page: a1644
 and Toshihisa Ohshima
Toshihisa OhshimaAffiliation: Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, Japan
For correspondence: ohshima@bme.oit.ac.jp
Bio-protocol author page: a1645
,  date: 9/5/2014, 3935 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1231.

[Abstract] With the exception of glycine, α-amino acids are optically active, and two optical isomers (L- and D-) of each amino acid can be formed. Recent developments of analytical techniques have revealed that several free D-amino acids such as D-aspartate, D-serine and D-alanine exist in many kinds of organism ...

A Conceptual Outline for Omics Experiments Using Bioinformatics Analogies

Authors: Prashanth Suravajhala
Prashanth SuravajhalaAffiliation 1: Bioinformatics Organization, Hudson, USA
Affiliation 2: Bioclues.org, Hyderabad, India
Affiliation 3: Bioclues.org, Roskilde, Denmark
Affiliation 4: Quantitative Systems Genetics group, University of Copenhagen, Grønnegårdsvej, Frederiksberg
For correspondence: prash@bioinformatics.org
Bio-protocol author page: a1963
 and Jeffrey W. Bizzaro
Jeffrey W. BizzaroAffiliation: Bioinformatics Organization, Hudson, USA
Bio-protocol author page: a1964
,  date: 2/5/2015, 3837 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1387.

[Abstract] Hypothetical proteins (HP) are those that are not characterized in the laboratory and so remain “orphaned” in genomic databases. In recent times there has been a lot of progress in characterizing HPs in the laboratory. Various methods, such as sequence capture and Next Generation Sequencing (NGS), have ...

Spindle Angle Measurements

Authors: Julien Cau
Julien CauAffiliation: IGH, CNRS UPR1142, Montpellier, France
For correspondence: julien.cau@igh.cnrs.fr
Bio-protocol author page: a895
, Nathalie Morin
Nathalie MorinAffiliation: CRBM, CNRS UMR5237, Universités Montpellier 1 et 2, Montpellier, France
Bio-protocol author page: a894
 and Guillaume Bompard
Guillaume BompardAffiliation: IGH, CNRS UPR1142, Montpellier, France
For correspondence: guillaume.bompard@igh.cnrs.fr
Bio-protocol author page: a890
,  date: 10/5/2013, 3813 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.925.

[Abstract] Spindle angles measures derive from the measures of spindle poles positions that were taken from fixed and immunostained adherent cells. To determine spindle angles (α), z-stack images of metaphasic cells immunostained with anti γ-tubulin (spindle poles) and anti β-tubulin antibodies (mitotic spindle) ...

Pulse Chase of Suspension Cells

Authors: Lai-Yee Wong
Lai-Yee WongAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
For correspondence: laiyee.wong@usc.edu
Bio-protocol author page: a1453
, QiMing Liang
QiMing LiangAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
Bio-protocol author page: a1454
, Kevin Brulois
Kevin BruloisAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
Bio-protocol author page: a1455
 and Jae Jung
Jae JungAffiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles, CA, USA
Bio-protocol author page: a1456
,  date: 7/5/2014, 3746 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1170.

[Abstract] Pulse-chase method is a powerful technique used to follow the dynamics of proteins over a period of time. The expression level, processing, transport, secretion or half-life of proteins can be tracked by metabolically labeling the cells, such as with radiolabeled amino acids (pulse step). This protocol ...

Dye-uptake Experiment through Connexin Hemichannels

Authors: Andrea Puhar
Andrea PuharAffiliation: Inserm U786 and Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France
For correspondence: andrea.puhar@pasteur.fr
Bio-protocol author page: a1611
 and Philippe J. Sansonetti
Philippe J. SansonettiAffiliation: Microbiologie et Maladies Infectieuses, Collège de France, Paris, France
Bio-protocol author page: a1612
,  date: 9/5/2014, 3674 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1221.

[Abstract] Connexins (Cxs) are integral membrane proteins of vertebrates that associate to form hexameric transmembrane channels, named hemichannels. Twenty-one Cx types have been described, which are named according to their molecular weight. Cxs are expressed in many cell types, e.g. epithelial cells, astrocytes ...

Hydroxyproline Assay Using NaBr/NaOCl

Author: Derek T. A. Lamport
Derek T. A. LamportAffiliation: School of Life Sciences, University of Sussex, Falmer, Brighton, UK
For correspondence: derekt.t.a.lamport@googlemail.com
Bio-protocol author page: a864
,  date: 10/5/2013, 3511 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.917.

[Abstract] Hydroxyproline (Hyp) is a major constituent of a relatively few proteins that are major structural components of the extracellular matrix and primary cell wall of animals and plants respectively. Significant amounts of the cyclic amino acids proline and hydroxyproline decrease polypeptide flexibility; ...

Observation of Chloroplast-actin Filaments in Leaves of Arabidopsis

Authors: Sam-Geun Kong
Sam-Geun KongAffiliation: Department of Biology, Kyushu University, Fukuoka, Japan
For correspondence: kong.samgeun@gmail.com
Bio-protocol author page: a1082
 and Masamitsu Wada
Masamitsu Wada Affiliation: Department of Biology, Kyushu University, Fukuoka, Japan
Bio-protocol author page: a1083
,  date: 12/20/2013, 3164 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1008.

[Abstract] Chloroplast-actin (cp-actin) filaments play a pivotal role in chloroplast photorelocation movement. This protocol describes observation of cp-actin filaments in intact palisade cells of Arabidopsis leaves (Kong et al., 2013). The live cell imaging of cp-actin filaments is taken on moving chloroplasts, ...

Purification and Crystallization of Chloromuconolactone Dehalogenase ClcF from Rhodococcus opacus 1CP

Authors: Christian Roth
Christian RothAffiliation: York Structural Biology Laboratory, University of York, York, UK
For correspondence: christian.roth@york.ac.uk
Bio-protocol author page: a1301
, Janosch Alexander D. Gröning
Janosch Alexander D. GröningAffiliation: Environmental Microbiology IÖZ, Technical University "Bergakademie" Freiberg, Freiberg, Germany
Bio-protocol author page: a1302
, Stefan Rudolf Kaschabek
Stefan Rudolf KaschabekAffiliation: Environmental Microbiology IÖZ, Technical University "Bergakademie" Freiberg, Freiberg, Germany
Bio-protocol author page: a1303
, Michael Schlömann
Michael SchlömannAffiliation: Environmental Microbiology IÖZ, Technical University "Bergakademie" Freiberg, Freiberg, Germany
Bio-protocol author page: a1304
 and Norbert Sträter
Norbert Sträter Affiliation: Center for Biotechnology and Biomedicine, University Leipzig, Leipzig, Germany
Bio-protocol author page: a1305
,  date: 4/20/2014, 3088 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1107.

[Abstract] The protocol describes the generation of variants of chloromuconolactone dehalogenase from Rhodococcus opacus (R. opacus) 1CP. ClcF is a multimeric protein, which catalyses the dechlorination of 5-chloromuconolactone to cis-dienelactone in the 3-chlorocatecholic acid degradation pathway. The protocol ...

Zonal Sedimentation Analysis on Sucrose Gradients

Author: Javier G. Magadán
Javier G. MagadánAffiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA
For correspondence: magadaj@niaid.nih.gov
Bio-protocol author page: a1285
,  date: 4/20/2014, 2990 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1100.

[Abstract] Zonal sedimentation analysis on sucrose gradients allows estimation of the molecular size of an individual protein or a protein complex by centrifugation at a constant speed under nondenaturing conditions. This method is particularly suitable for globular proteins like the influenza A virus (IAV) protein ...

Fluorometric Estimation of Viral Thermal Stability

Authors: Vamseedhar Rayaprolu
Vamseedhar RayaproluAffiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, USA
Bio-protocol author page: a1553
, Shannon Kruse
Shannon KruseAffiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, USA
Bio-protocol author page: a1554
, Ravi Kant
Ravi KantAffiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, USA
Bio-protocol author page: a1555
, Navid Movahed
Navid MovahedAffiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, USA
Bio-protocol author page: a1556
, Dewey Brooke
Dewey BrookeAffiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, USA
Bio-protocol author page: a1557
 and Brian Bothner
Brian BothnerAffiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, USA
For correspondence: bbothner@chemistry.montana.edu
Bio-protocol author page: a1558
,  date: 8/5/2014, 2839 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1199.

[Abstract] Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly ...
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