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Biochemistry

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 ...

Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies

Authors: Megan Sjodt
Megan SjodtAffiliation 1: Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA
Affiliation 2: Department of Chemistry and Biochemistry, UCLA-DOE Institute of Genomics and Proteomics and Molecular Biology Institute, University of California, Los Angeles, USA
Bio-protocol author page: a4249
 and Robert T. Clubb
Robert T. ClubbAffiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute of Genomics and Proteomics and Molecular Biology Institute, University of California, Los Angeles, USA
For correspondence: rclubb@mbi.ucla.edu
Bio-protocol author page: a4250
,  date: 4/5/2017, 554 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2207.

[Abstract] Site-specific attachment of paramagnetic spin labels to biomolecules causes distance-dependent line-broadening effects, which can be exploited to study the structure and dynamics of these molecules in solution. This protocol describes how to attach nitroxide spin labels to proteins and how to collect and analyze NMR data using these labeled samples. ...

Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy

Authors: Yuxiang Jiang
Yuxiang JiangAffiliation 1: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China
Affiliation 2: Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4178
 and Shanjin Huang
Shanjin HuangAffiliation 1: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China
Affiliation 2: Institute of Botany, Chinese Academy of Sciences, Beijing, China
For correspondence: sjhuang@tsinghua.edu.cn
Bio-protocol author page: a966
,  date: 3/5/2017, 739 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2146.

[Abstract] Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, fragmentation ...

Tandem Purification of His6-3x FLAG Tagged Proteins for Mass Spectrometry from Arabidopsis

Authors: He Huang
He Huang Affiliation: Donald Danforth Plant Science Center, St. Louis, USA
Bio-protocol author page: a3873
 and Dmitri Anton Nusinow
Dmitri Anton NusinowAffiliation: Donald Danforth Plant Science Center, St. Louis, USA
For correspondence: meter@danforthcenter.org
Bio-protocol author page: a3874
,  date: 12/5/2016, 1652 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2060.

[Abstract] Tandem affinity purification is a powerful method to identify protein complexes that function in association with a known gene of interest. This protocol describes a methodology to capture proteins tagged with His6-3x FLAG explicitly for the purpose of on-bead digestion and identification by mass spectrometry. The high sensitivity and specificity of ...

Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)

Authors: Michael J. Munson
Michael J. MunsonAffiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK
Bio-protocol author page: a3428
 and Ian G. Ganley
Ian G. GanleyAffiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK
For correspondence: i.ganley@dundee.ac.uk
Bio-protocol author page: a3429
,  date: 8/20/2016, 1252 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1903.

[Abstract] The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately ...

Micro-scale NMR Experiments for Monitoring the Optimization of Membrane Protein Solutions for Structural Biology

Authors: Reto Horst
Reto HorstAffiliation: Structural Biology and Biophysics Group, Pfizer Worldwide Research and Development, Eastern Point Road, Groton, USA
Bio-protocol author page: a2396
 and Kurt Wüthrich
Kurt WüthrichAffiliation: Department of Integrative Structural and Computational Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, USA
For correspondence: wuthrich@scripps.edu
Bio-protocol author page: a2397
,  date: 7/20/2015, 1705 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1539.

[Abstract] Reconstitution of integral membrane proteins (IMP) in aqueous solutions of detergent micelles has been extensively used in structural biology, using either X-ray crystallography or NMR in solution. Further progress could be achieved by establishing a rational basis for the selection of detergent and buffer conditions, since the stringent bottleneck ...

Biotinylation and Purification of Surface-exposed Helicobacter pylori Proteins

Authors: Bradley J. Voss
Bradley J. VossAffiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, USA
Bio-protocol author page: a2157
 and Timothy L. Cover
Timothy L. CoverAffiliation 1: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, USA
Affiliation 2: Division of Infectious Diseases, Medical Center North, Vanderbilt University School of Medicine, Nashville, USA
Affiliation 3: Veterans Affairs Tennessee Valley Healthcare System, Vanderbilt University School of Medicine, Nashville, USA
Affiliation 4: Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University School of Medicine, Nashville, USA
For correspondence: timothy.l.cover@vanderbilt.edu
Bio-protocol author page: a2158
,  date: 4/20/2015, 2186 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1455.

[Abstract] Interactions between pathogenic bacteria and host cells are often mediated by proteins found on the surfaces of the bacteria. The Gram-negative bacterium Helicobacter pylori is predicted to produce at least 50 surface-exposed outer membrane proteins, but there has been relatively little progress in experimentally analyzing the cell-surface proteome ...

Detection of the Secreted and Cytoplasmic Fractions of IpaB, IpaC and IpaD by Lysozyme Permeabilization

Authors: François Xavier Campbell-Valois
François Xavier Campbell-ValoisAffiliation 1: Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France
Affiliation 2: INSERM, Paris, France
For correspondence: fxcamval@pasteur.fr
Bio-protocol author page: a1738
, Pamela Schnupf
Pamela SchnupfAffiliation 1: Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France
Affiliation 2: INSERM, Paris, France
Bio-protocol author page: a1739
 and Philippe J. Sansonetti
Philippe J. SansonettiAffiliation 1: Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France
Affiliation 2: INSERM, Paris, France
Affiliation 3: Collège de France, Chaire de Microbiologie et Maladies infectieuses, Paris, France
Bio-protocol author page: a1612
,  date: 10/20/2014, 2770 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1271.

[Abstract] Gram negative bacterial pathogens, such as Shigella flexneri, which possess a Type Three Secretion System (T3SS), are able to transfer bacterial proteins, dubbed translocators and effectors, from their cytoplasm into the cytoplasm of their host cells using a syringe like needle complex. For Shigella, it has been shown that during cellular invasion, ...

Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans

Authors: Takuji Oka
Takuji OkaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
For correspondence: oka@bio.sojo-u.ac.jp
Bio-protocol author page: a1613
, Yukako Katafuchi
Yukako KatafuchiAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1614
, Kohsai Fukuda
Kohsai FukudaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1615
, Keisuke Ekino
Keisuke EkinoAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1616
, Masatoshi Goto
Masatoshi GotoAffiliation: Department of Bioscience and Biotechnology, Kyushu University, Fukuoka City, Japan
Bio-protocol author page: a1617
 and Yoshiyuki Nomura
Yoshiyuki NomuraAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1618
,  date: 9/5/2014, 2647 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1222.

[Abstract] GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces ...

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 describes the condition used to study the folding ...
1 2 

Protein Translation Study – Label Protein with S35 Methionine in Cells

Authors: Salma Hasan
Salma HasanAffiliation: INSERM U1009, Gustave Roussy, Villejuif, France
Bio-protocol author page: a140
 and Isabelle Plo
Isabelle PloAffiliation: INSERM U1009, Gustave Roussy, Villejuif, France
For correspondence: isabelle.plo@gustaveroussy.fr
Bio-protocol author page: a141
,  date: 11/5/2012, 11999 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.282.

[Abstract] To follow protein synthesis, cells should be incubated with radioactive amino acid such as [35S] methionine during mRNA translation. Then, the neosynthetized protein will be identified by an autoradiography after immunoprecipitation with a specific antibody and separation on a polyacrylamide denaturing ...

Cell Surface Protein Biotinylation and Analysis

Authors: Anna Tarradas
Anna TarradasAffiliation: Cardiovascular Genetics Center, Girona Biomedical Research Institute (IDIBGI) & Univerisity of Girona, Girona, Spain
Bio-protocol author page: a750
, Elisabet Selga
Elisabet SelgaAffiliation: Cardiovascular Genetics Center & Medical Sciences Department, Girona Biomedical Research Institute (IDIBGI) & University of Girona, Girona, Spain
Bio-protocol author page: a751
, Helena Riuró
Helena RiuróAffiliation: Cardiovascular Genetics Center, Girona Biomedical Research Institute (IDIBGI), Girona, Spain
Bio-protocol author page: a752
, Fabiana S. Scornik
Fabiana S. ScornikAffiliation: Cardiovascular Genetics Center & Medical Sciences Department, Girona Biomedical Research Institute (IDIBGI) & Univ. of Girona School of Medicine, Girona, Spain
Bio-protocol author page: a753
, Ramon Brugada
Ramon BrugadaAffiliation: Cardiovascular Genetics Center & Medical Sciences Department, Girona Biomedical Research Institute (IDIBGI) & Univerisity of Girona School of Medicine, Girona, Spain
Bio-protocol author page: a754
 and Marcel Vergés
Marcel VergésAffiliation: Cardiovascular Genetics Center & Medical Sciences Department, Girona Biomedical Research Institute (IDIBGI) & Univerisity of Girona School of Medicine, Girona, Spain
For correspondence: mverges@gencardio.com
Bio-protocol author page: a755
,  date: 8/20/2013, 7167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.857.

[Abstract] A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, ...

[Bio101] Metabolic Labeling of Yeast Proteins

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, 6738 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.13.

[Abstract] Proteins of Saccharomyces cerevisiae can be metabolically labeled with (35) methionine. After labeling, a protocol is described for the mechanical disruption of yeast cells or conversion to spheroplasts, with subsequent lysis before immunoprecipitation of the proteins....

[Bio101] Labeling Protein with Thiol-reactive Probes

Author: Qingrong Yan,  date: 6/5/2011, 5313 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.82.

[Abstract] This protocol is used to label protein or peptide with a maleinmide or iodoacetamide conjugated fluorescent probe through the free cysteine....

LC3B Labeling on Terrestrial Isopod Adipocytes

Authors: Christine Braquart-Varnier
Christine Braquart-VarnierAffiliation: Ecologie et Biologie des Interactions, UMR 7267 CNRS, Université de Poitiers, Poitiers Cedex, France
For correspondence: christine.braquart@univ-poitiers.fr
Bio-protocol author page: a625
, Maryline Raimond
Maryline RaimondAffiliation: Ecologie et Biologie des Interactions, UMR 7267 CNRS, Université de Poitiers, Poitiers Cedex, France
Bio-protocol author page: a626
 and Mathieu Sicard
Mathieu SicardAffiliation: Ecologie et Biologie des Interactions, UMR 7267 CNRS, Université de Poitiers, Poitiers Cedex, France
Bio-protocol author page: a341
,  date: 6/20/2013, 4042 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.792.

[Abstract] The LC3B protein plays a critical role in autophagy. Normally, this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the phagophore. This localization can be used as a general marker for autophagic membranes. To visualize the LC3B, ...

35S pulse Labelling of Chlamydomonas Chloroplast Proteins

Authors: Alexandra-Viola Bohne
Alexandra-Viola BohneAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
Bio-protocol author page: a615
, Christian Schwarz
Christian SchwarzAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
Bio-protocol author page: a609
 and Joerg Nickelsen
Joerg NickelsenAffiliation: Molecular Plant Science, Ludwig-Maximilians-Universität, Munich, Germany
For correspondence: joerg.nickelsen@lrz.uni-muenchen.de
Bio-protocol author page: a610
,  date: 6/5/2013, 3973 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.783.

[Abstract] 35S pulse labelling of proteins is used to attach a radioactive label to newly synthesized proteins, as sulfur is an element that is mainly present in proteins (Fleischmann and Rochaix 1999). Depending on your organism’s uptake mechanisms you need cysteine, methionine or sulfuric acid as a source of ...

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 ...

Detection of the Secreted and Cytoplasmic Fractions of IpaB, IpaC and IpaD by Lysozyme Permeabilization

Authors: François Xavier Campbell-Valois
François Xavier Campbell-ValoisAffiliation 1: Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France
Affiliation 2: INSERM, Paris, France
For correspondence: fxcamval@pasteur.fr
Bio-protocol author page: a1738
, Pamela Schnupf
Pamela SchnupfAffiliation 1: Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France
Affiliation 2: INSERM, Paris, France
Bio-protocol author page: a1739
 and Philippe J. Sansonetti
Philippe J. SansonettiAffiliation 1: Institut Pasteur, Unité de Pathogénie Microbienne Moléculaire, Paris, France
Affiliation 2: INSERM, Paris, France
Affiliation 3: Collège de France, Chaire de Microbiologie et Maladies infectieuses, Paris, France
Bio-protocol author page: a1612
,  date: 10/20/2014, 2770 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1271.

[Abstract] Gram negative bacterial pathogens, such as Shigella flexneri, which possess a Type Three Secretion System (T3SS), are able to transfer bacterial proteins, dubbed translocators and effectors, from their cytoplasm into the cytoplasm of their host cells using a syringe like needle complex. For Shigella, ...

Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans

Authors: Takuji Oka
Takuji OkaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
For correspondence: oka@bio.sojo-u.ac.jp
Bio-protocol author page: a1613
, Yukako Katafuchi
Yukako KatafuchiAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1614
, Kohsai Fukuda
Kohsai FukudaAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1615
, Keisuke Ekino
Keisuke EkinoAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1616
, Masatoshi Goto
Masatoshi GotoAffiliation: Department of Bioscience and Biotechnology, Kyushu University, Fukuoka City, Japan
Bio-protocol author page: a1617
 and Yoshiyuki Nomura
Yoshiyuki NomuraAffiliation: Department of Applied Microbial Technology, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a1618
,  date: 9/5/2014, 2647 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1222.

[Abstract] GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in ...

Biotinylation and Purification of Surface-exposed Helicobacter pylori Proteins

Authors: Bradley J. Voss
Bradley J. VossAffiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, USA
Bio-protocol author page: a2157
 and Timothy L. Cover
Timothy L. CoverAffiliation 1: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, USA
Affiliation 2: Division of Infectious Diseases, Medical Center North, Vanderbilt University School of Medicine, Nashville, USA
Affiliation 3: Veterans Affairs Tennessee Valley Healthcare System, Vanderbilt University School of Medicine, Nashville, USA
Affiliation 4: Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt University School of Medicine, Nashville, USA
For correspondence: timothy.l.cover@vanderbilt.edu
Bio-protocol author page: a2158
,  date: 4/20/2015, 2186 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1455.

[Abstract] Interactions between pathogenic bacteria and host cells are often mediated by proteins found on the surfaces of the bacteria. The Gram-negative bacterium Helicobacter pylori is predicted to produce at least 50 surface-exposed outer membrane proteins, but there has been relatively little progress in ...
1 2 
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