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Measuring Oxygen Consumption Rate in Caenorhabditis elegans

Featured protocol,  Authors: Konstantinos Palikaras
Konstantinos PalikarasAffiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Bio-protocol author page: a3832
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 12/5/2016, 44 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2049.

Brief version appeared in Nature, May 2015
The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating the switch from oxidative phosphorylation to aerobic glycolysis (Chen et al., 2015; Vander Heiden et al., 2009). In this protocol, we describe a method for the determination of oxygen consumption rates in the nematode Caenorhabditis elegans.

Intracellular Assessment of ATP Levels in Caenorhabditis elegans

Featured protocol,  Authors: Konstantinos Palikaras
Konstantinos PalikarasAffiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Bio-protocol author page: a3832
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2048.

Brief version appeared in Nature, May 2015
Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans.

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Featured protocol,  Authors: Beth Mann
Beth MannAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3796
Lip Nam Loh
Lip Nam LohAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3797
Geli Gao
Geli GaoAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3798
 and Elaine Tuomanen
Elaine TuomanenAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
For correspondence: Elaine.tuomanen@stjude.org
Bio-protocol author page: a3799
date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2037.

Brief version appeared in Cell Host Microbe, Mar 2016
Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. This protocol describes how to prepare purified cell wall from Streptococcus pneumoniae, detect its distribution in animal tissues, and study the tissue response using the placenta and fetal brain as examples.

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

Featured protocol,  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, 192 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2060.

Brief version appeared in eLife, Feb 2016
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 our methods allow for purification of proteins expressed at native levels from endogenous promoters to enable uncovering the functional roles of plant protein complexes.

Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves

Featured protocol,  Authors: Masaki Okumura
Masaki OkumuraAffiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Bio-protocol author page: a3819
 and Toshinori Kinoshita
Toshinori KinoshitaAffiliation 1: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Affiliation 2: Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan
For correspondence: kinoshita@bio.nagoya-u.ac.jp
Bio-protocol author page: a3820
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2044.

Brief version appeared in Plant Physiol, May 2016
Plant plasma membrane H+-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H+ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic activity of the plasma membrane H+-ATPase from Arabidopsis thaliana leaves.

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation

Featured protocol,  Authors: Roberta Antonelli
Roberta AntonelliAffiliation 1: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Affiliation 2: Laboratory of Translational Research in Child and Adolescent Cancer, Vall d'Hebron Research Institute (VHIR)-UAB, Barcelona, Spain
For correspondence: roberta.antonelli@vhir.org
Bio-protocol author page: a3828
 and Paola Zacchi
Paola ZacchiAffiliation: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Bio-protocol author page: a3829
date: 12/5/2016, 26 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2046.

Brief version appeared in J Neurosci, May 2016
Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex with it or to detect post-translational modifications. The mitotic protein monoclonal 2 (MPM-2) is an antibody originally raised against extracts of synchronized mitotic HeLa cells to identify proteins selectively present in mitotic, and not in interphase-cells (Davis et al., 1983). MPM-2 recognizes phosphorylated serine or threonine residues followed by proline (pS/T-P), consensus epitopes generated by the concerted action of proline-directed protein kinases and phosphatases (Lu et al., 2002). These reversible phosphorylation events have emerged to control various cellular processes by promoting conformational changes on the target that are not simply due to the phosphorylation event per se. These motifs, once phosphorylated, are able to recruit Pin1 (Peptidyl-prolyl Isomerase NIMA interacting protein 1) (Lu et al., 1996; Lu and Zhou, 2007), a chaperone which drives the cis/trans isomerization reaction on the peptide bond, switching the substrate between functionally diverse conformations (Lu, 2004; Wulf et al., 2005). This protocol describes a general MPM-2 based immunoprecipitation strategy using the scaffolding molecule postsynaptic density protein-95 (PSD-95) (Chen et al., 2005), a neuronal Pin1 target (Antonelli et al., 2016), as an example to illustrate the detailed procedure.

Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae

Featured protocol,  Authors: Yuki Kobayashi
Yuki KobayashiAffiliation: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Bio-protocol author page: a3789
 and Kan Tanaka
Kan TanakaAffiliation 1: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Affiliation 2: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Saitama, Japan
For correspondence: kntanaka@res.titech.ac.jp
Bio-protocol author page: a3790
date: 12/5/2016, 35 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2033.

Brief version appeared in Plant Cell Physiol, May 2016
Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance (Kobayashi et al., 2016). This report describes the detailed method for the extraction and quantification of ABA in the model red alga C. merolae.

Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria

Featured protocol,  Authors: Monica Benincasa*
Monica BenincasaAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3800
Quentin Barrière*
Quentin BarrièreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3801
Giulia Runti
Giulia RuntiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3802
Olivier Pierre
Olivier PierreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3803
Mick Bourge
Mick BourgeAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3804
Marco Scocchi
Marco ScocchiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
For correspondence: mscocchi@units.it
Bio-protocol author page: a3805
 and Peter Mergaert
Peter MergaertAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
For correspondence: peter.mergaert@i2bc.paris-saclay.fr
Bio-protocol author page: a3806
 (*contributed equally to this work) date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2038.

Brief version appeared in MPMI, Nov 2015
Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher trypan blue. Trypan blue is excluded from the interior of intact cells and the fluorescence of the extracellular peptide or of the peptide bound on the bacterial surface can be quenched by it, while the fluorescence of the internalized peptide is not affected. The uptake of the peptide by the bacteria is determined by measuring the fluorescence in individual cells by flow cytometry.

Protocol for Molecular Dynamics Simulations of Proteins

Featured protocol,  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, 130 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2051.

Brief version appeared in BMC Genomics, Oct 2013
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.

Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes

Featured protocol,  Authors: Mariana Edith Tellechea*
Mariana Edith TellecheaAffiliation 1: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari,Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Affiliation 2: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3780
Javier Garcia-Pardo*
Javier Garcia-PardoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3781
Juliana Cotabarren
Juliana CotabarrenAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3782
Daniela Lufrano
Daniela LufranoAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3783
Laura Bakas
Laura BakasAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3784
Francesc Xavier Avilés
Francesc Xavier AvilésAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3785
Walter David Obregon
Walter David ObregonAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3786
Julia Lorenzo
Julia LorenzoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
For correspondence: julia.lorenzo@uab.es
Bio-protocol author page: a3787
 and Sebastián Tanco
Sebastián TancoAffiliation 1: Medical Biotechnology Center, VIB, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
For correspondence: sebastian.tanco@vib-ugent.be
Bio-protocol author page: a3788
 (*contributed equally to this work) date: 12/5/2016, 40 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2032.

Brief version appeared in Phytochemistry Dec 2015
Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of new MCP inhibitors from plants constitute a valuable approach for the development of new therapeutic strategies. Herein we describe a simple and accessible microplate method for the study of the specific and dose-response carboxypeptidase A inhibitory activities present in Andean potato tubers. Our protocol combines an extraction method optimized for small protein inhibitors in plant tissues, with the measurement of enzyme kinetics using a microplate reader. These instruments are capable of reading small sample volumes, for many samples in a very short time-frame, therefore reducing the time and costs of high-throughput screening experiments. Although this protocol describes the study of Andean potatoes, our approach is also applicable to the analysis other plant samples.

Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression

Featured protocol,  Authors: Jennifer L. Larson-Casey
Jennifer L. Larson-CaseyAffiliation: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Bio-protocol author page: a3716
 and A. Brent Carter
A. Brent CarterAffiliation 1: Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Affiliation 2: Birmingham Veterans Administration Medical Center, Birmingham, AL, USA
For correspondence: bcarter1@uab.edu
Bio-protocol author page: a3717
date: 11/20/2016, 152 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2009.

Brief version appeared in Immunity, Mar 2016
Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).

Heterologous Expression and Purification of the Magnesium Transporter A (MgtA) in Escherichia coli

Featured protocol,  Authors: Saranya Subramani
Saranya SubramaniAffiliation: Norwegian Centre for Molecular Medicine, Nordic EMBL Partnership University of Oslo, Oslo, Norway
Bio-protocol author page: a3695
 and Jens Preben Morth
Jens Preben MorthAffiliation 1: Norwegian Centre for Molecular Medicine, Nordic EMBL Partnership University of Oslo, Oslo, Norway
Affiliation 2: Institute for Experimental Medical Research, Oslo University Hospital, Oslo, Norway
For correspondence: j.p.morth@ncmm.uio.no
Bio-protocol author page: a3696
date: 11/20/2016, 148 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2001.

Brief version appeared in Elife, Jan 2016
The magnesium transporter A (MgtA) is a magnesium transporting P-type ATPase present in prokaryotes and plants (Subramani et al., 2016). In Salmonella typhimurium and Escherichia coli (E. coli), MgtA is expressed only in magnesium limiting conditions and plays an important role in Mg2+ homeostasis (Groisman et al., 2013). The transcription of mgtA is regulated by the two-component system PhoP/PhoQ (Soncini et al., 1996; Kato et al., 1999). The membrane bound histidine kinase, PhoQ, senses low Mg2+ concentration in the periplasmic space and phosphorylates its cognate response regulator, PhoP, which initiates mgtA transcription (Groisman et al., 2013). MgtA is targeted to the plasma membrane and facilitate the bacterial survival under low Mg2+ condition, by importing Mg2+ into the cytoplasm. The MgtA homolog in petunia (PH1) is found in the vacuolar membrane and involved with the coloration of the flower petals (Faraco et al., 2014). As a first step towards understanding the molecular details of MgtA Mg2+ transport, we describe a detailed protocol for the purification of E. coli MgtA that can be used for biochemical and biophysical studies. Recombinant E. coli MgtA with hexa histidine tag at the N-terminus was cloned from E. coli DH5α and over expressed in the E. coli C43(DE3) by fermentation to an OD > 6. Cell lysis was performed in a high pressure homogenizer and the membranes were isolated by ultracentrifugation. Membrane proteins were solubilized with the detergent dodecyl-β-D maltoside. MgtA was purified by affinity and size exclusion chromatography. Final yields of purified MgtA reach ~1 mg MgtA per 3 g of wet cell pellet.

Total Histone Acid Extraction of Colon Cancer HCT116 Cells

Featured protocol,  Authors: Lin-Lin Cao
Lin-Lin CaoAffiliation: Department of Clinical Laboratory, Peking University People’s Hospital, Beijing, China
Bio-protocol author page: a3751
 and Wei-Guo Zhu
Wei-Guo ZhuAffiliation 1: Department of Biochemistry and Molecular Biology, School of Medicine, Shenzhen University, Shenzhen, China
Affiliation 2: Peking University-Tsinghua University Joint Center for Life Sciences, Beijing, China
For correspondence: zhuweiguo@szu.edu.cn
Bio-protocol author page: a3752
date: 11/20/2016, 117 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2023.

Brief version appeared in Oncogene, Jan 2016
Histone acid extraction assay is a popular method to determine histone modification levels in mammalian cells. It includes three steps: first, histones are released from chromatin by sulfuric acid; trichloroacetate (TCA) is then added to precipitate histones; and finally, histones are dissolved in double-distilled H2O (ddH2O). Here we present a detailed histone acid extraction assay in our laboratory using a colon cancer cell line, HCT116, as a model.

Ionization Properties of Phospholipids Determined by Zeta Potential Measurements

Featured protocol,  Authors: Murugappan Sathappa
Murugappan SathappaAffiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA
Bio-protocol author page: a3772
 and Nathan N. Alder
Nathan N. AlderAffiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA
For correspondence: nathan.alder@uconn.edu
Bio-protocol author page: a3773
date: 11/20/2016, 113 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2030.

Brief version appeared in Biochim Biophys Acta, Jun 2016
Biological membranes are vital for diverse cellular functions such as maintaining cell and organelle structure, selective permeability, active transport, and signaling. The surface charge of the membrane bilayer plays a critical role in these myriad processes. For most biomembranes, the surface charge of anionic phospholipids contributes to the negative surface charge density within the interfacial region of the bilayer. To quantify surface charge, it is essential to understand the proton dissociation behavior of the titratable headgroups within such lipids. We describe a protocol that uses model membranes for electrokinetic zeta potential measurements coupled with data analysis using Gouy-Chapman-Stern formalism to determine the pKa value of the component lipids. A detailed example is provided for homogeneous bilayers composed of the monoanionic lipid phosphatidylglycerol. This approach can be adapted for the measurement of bilayers with a heterogeneous lipid combination, as well as for lipids with multiple titratable sites in the headgroup (e.g., cardiolipin).

Determining Efficiency and Selectivity of Lipid Extraction by Perturbing Agents from Model Membranes

Featured protocol,  Authors: Michel Lafleur
Michel LafleurAffiliation: Department of chemistry, Université de Montréal, Montréal (Québec), Canada
For correspondence: michel.lafleur@umontreal.ca
Bio-protocol author page: a3731
 and Alexandre Therrien
Alexandre TherrienAffiliation: Department of chemistry, Université de Montréal, Montréal (Québec), Canada
Bio-protocol author page: a3732
date: 11/20/2016, 113 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2016.

Brief version appeared in J Phys Chem B, May 2016
Several membrane-perturbing agents extract lipids from membranes and, in some cases, this lipid efflux is lipid specific. In order to gain a better description of this phenomenon and to detail the intermolecular interactions that are involved, a method has been developed to characterize the extent and the specificity of membrane-lipid extraction by perturbing agents. A perturbing agent is incubated with model membranes existing as multilamellar vesicles (MLVs) and subsequently, the remaining MLVs and the small lipid/perturbing agent complexes resulting from the extraction are isolated and analysed to assess the extent and the specificity of the lipid extraction.

Determination of Rate of [3H-methyl]-choline Incorporation into Cellular Lipids and Non-lipid Metabolites

Featured protocol,  Authors: Tim Andrew Davies Smith
Tim Andrew Davies SmithAffiliation: School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, Aberdeen, UK
For correspondence: t.smith@abdn.ac.uk
Bio-protocol author page: a3739
 and Su Myat Phyu
Su Myat PhyuAffiliation: School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, Aberdeen, UK
Bio-protocol author page: a3740
date: 11/20/2016, 113 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2019.

Brief version appeared in PLoS One, Mar 2016
The choline-containing phospholipid, phosphatidylcholine (PtdCho) is the most common mammalian phospholipid found in cell membrane (Ide et al., 2013). It is also a component of intracellular signalling pathways (Cui and Houweling, 2002). Herein is described a measure of the rate of accumulation of choline by lipid soluble PtdCho and lyso-Ptdcho which can further be discriminated by chromatographic analysis (Smith and Phyu, 2016). Determination of the accumulation of [3H-methyl]-choline into water-soluble components is also described. The procedure could be used to measure the effect of drugs and other factors on choline incorporation into phospholipids. After exposure of cells to test conditions (e.g., drugs) adherent cells in tissue culture flasks are incubated with radiolabelled [3H-methyl]-choline in medium for 15 min (pulse). The [3H-methyl]-choline is then rapidly removed and incubation continued in the presence of non-radioactive medium (chase). Cellular distribution of [3H-methyl] is then determined by cell fractionation and measurement of radioactivity in the lipid and non-lipid cellular components.

Measuring Oxygen Consumption Rate in Caenorhabditis elegans

Authors: Konstantinos Palikaras
Konstantinos PalikarasAffiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Bio-protocol author page: a3832
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 12/5/2016, 44 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2049.

[Abstract] The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating the switch from oxidative phosphorylation ...

Intracellular Assessment of ATP Levels in Caenorhabditis elegans

Authors: Konstantinos Palikaras
Konstantinos PalikarasAffiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Bio-protocol author page: a3832
 and Nektarios Tavernarakis
Nektarios TavernarakisAffiliation 1: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Crete, Greece
Affiliation 2: Department of Basic Sciences, Faculty of Medicine, University of Crete, Crete, Greece
For correspondence: tavernarakis@imbb.forth.gr
Bio-protocol author page: a3833
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2048.

[Abstract] Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels ...

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Authors: Beth Mann
Beth MannAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3796
Lip Nam Loh
Lip Nam LohAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3797
Geli Gao
Geli GaoAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3798
 and Elaine Tuomanen
Elaine TuomanenAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
For correspondence: Elaine.tuomanen@stjude.org
Bio-protocol author page: a3799
date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2037.

[Abstract] Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. ...

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, 192 views, 0 Q&A, How to cite
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 ...

Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves

Authors: Masaki Okumura
Masaki OkumuraAffiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Bio-protocol author page: a3819
 and Toshinori Kinoshita
Toshinori KinoshitaAffiliation 1: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan
Affiliation 2: Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan
For correspondence: kinoshita@bio.nagoya-u.ac.jp
Bio-protocol author page: a3820
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2044.

[Abstract] Plant plasma membrane H+-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H+ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic ...

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation

Authors: Roberta Antonelli
Roberta AntonelliAffiliation 1: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Affiliation 2: Laboratory of Translational Research in Child and Adolescent Cancer, Vall d'Hebron Research Institute (VHIR)-UAB, Barcelona, Spain
For correspondence: roberta.antonelli@vhir.org
Bio-protocol author page: a3828
 and Paola Zacchi
Paola ZacchiAffiliation: International School for Advanced Studies, Neurobiology Department, Trieste, Italy
Bio-protocol author page: a3829
date: 12/5/2016, 26 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2046.

[Abstract] Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex ...

Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae

Authors: Yuki Kobayashi
Yuki KobayashiAffiliation: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Bio-protocol author page: a3789
 and Kan Tanaka
Kan TanakaAffiliation 1: Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan
Affiliation 2: Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Saitama, Japan
For correspondence: kntanaka@res.titech.ac.jp
Bio-protocol author page: a3790
date: 12/5/2016, 35 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2033.

[Abstract] Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance ...

Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria

Authors: Monica Benincasa*
Monica BenincasaAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3800
Quentin Barrière*
Quentin BarrièreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3801
Giulia Runti
Giulia RuntiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3802
Olivier Pierre
Olivier PierreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3803
Mick Bourge
Mick BourgeAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3804
Marco Scocchi
Marco ScocchiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
For correspondence: mscocchi@units.it
Bio-protocol author page: a3805
 and Peter Mergaert
Peter MergaertAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
For correspondence: peter.mergaert@i2bc.paris-saclay.fr
Bio-protocol author page: a3806
 (*contributed equally to this work) date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2038.

[Abstract] Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher trypan ...

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, 130 views, 0 Q&A, How to cite
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.
Keywords: Molecular dynamics simulations, Conformational ...

Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes

Authors: Mariana Edith Tellechea*
Mariana Edith TellecheaAffiliation 1: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari,Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Affiliation 2: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3780
Javier Garcia-Pardo*
Javier Garcia-PardoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3781
Juliana Cotabarren
Juliana CotabarrenAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3782
Daniela Lufrano
Daniela LufranoAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3783
Laura Bakas
Laura BakasAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3784
Francesc Xavier Avilés
Francesc Xavier AvilésAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Bio-protocol author page: a3785
Walter David Obregon
Walter David ObregonAffiliation: Centro de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
Bio-protocol author page: a3786
Julia Lorenzo
Julia LorenzoAffiliation: Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Campus Universitari, Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
For correspondence: julia.lorenzo@uab.es
Bio-protocol author page: a3787
 and Sebastián Tanco
Sebastián TancoAffiliation 1: Medical Biotechnology Center, VIB, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
For correspondence: sebastian.tanco@vib-ugent.be
Bio-protocol author page: a3788
 (*contributed equally to this work) date: 12/5/2016, 40 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2032.

[Abstract] Metallocarboxypeptidases (MCP) are zinc-dependent exopeptidases that catalyze the hydrolysis of C-terminal amide bonds in proteins and peptides. They are involved in a wide range of physiological processes and have recently emerged as relevant drug targets in biomedicine (Arolas et al., 2007). In this context, the study and discovery of new MCP inhibitors ...
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[Bio101] Bradford Protein Assay

Author: Fanglian He date: 3/20/2011, 71415 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.45.

[Abstract] The Bradford protein assay is used to measure the concentration of total protein in a sample. The principle of this assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a color change from brown to blue. This method actually measures the presence of the ...

[Bio101] Laemmli-SDS-PAGE

Author: Fanglian He date: 6/5/2011, 58181 views, 10 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.80.

[Abstract] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate proteins with relative molecular mass no smaller than 10 KD. Very small proteins (<10 KD) are difficult to resolve due to low ability of binding to SDS, which can be solved by gradient gels or using different eletrophoresis ...

[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, 38134 views, 4 Q&A, How to cite
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....

Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA)

Authors: Rabii Ameziane-El-Hassani
Rabii Ameziane-El-HassaniAffiliation 1: UBRM, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, Rabat, Morocco
Affiliation 2: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Bio-protocol author page: a200
 and Corinne Dupuy
Corinne DupuyAffiliation 1: Institut Gustave Roussy, FRE2939 Centre National de la Recherche Scientifique, Villejuif, France
Affiliation 2: University Paris, Sud 11, Orsay, France
For correspondence: dupuy@igr.fr
Bio-protocol author page: a201
date: 1/5/2013, 36844 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.313.

[Abstract] Reactive oxygen species (ROS) play a critical role in cellular physiopathology. ROS are implicated in cell proliferation, signaling pathways, oxidative defense mechanisms responsible for killing of bacteria, thyroid hormonosynthesis, etc. The cellular Redox homeostasis is balanced by oxidants and antioxidants ...

Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves Updates
The author made some updates (highlighted in blue) to the protocol on 09/19/2016.

Authors: Arsalan Daudi
Arsalan DaudiAffiliation 1: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Affiliation 2: Department of Plant Pathology, University of California, Davis, CA, USA
For correspondence: aadaudi@ucdavis.edu
Bio-protocol author page: a107
 and Jose A. O’Brien
Jose A. O’BrienAffiliation: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Bio-protocol author page: a108
date: 9/20/2012, 29506 views, 15 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.263.

[Abstract] In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi ...

[Bio101] Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/20/2011, 29068 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.95.

[Abstract] Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, ...

[Bio101] BCA (Bicinchoninic Acid) Protein Assay

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fhe@bio-protocol.org
Bio-protocol author page: a9
date: 3/5/2011, 28701 views, 3 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.44.

[Abstract] The BCA protein assay is used for quantitation of total protein in a sample. The principle of this method is that proteins can reduce Cu+2 to Cu+1 in an alkaline solution (the biuret reaction) and result in a purple color formation by bicinchoninic acid. The reduction of copper is mainly caused by four ...

[Bio101] Coomassie Blue Staining

Author: Fanglian He date: 6/5/2011, 27557 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.78.

[Abstract] Coomassie staining is able to detect protein bands containing about 0.2 μg or more protein. For low abundant protein, silver staining (www/silver staining) is a better choice since it is about 100-fold more sensitive than Coomassie staining....

[Bio101] Glucose Tolerance Test in Mice

Author: Peichuan Zhang
Peichuan ZhangAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Department of Biochemistry and Biophysics, University of California, San Francisco, USA
For correspondence: peichuan.zhang@ucsf.edu
Bio-protocol author page: a11
date: 10/5/2011, 27551 views, 4 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.159.

[Abstract] Glucose tolerance test is a standard procedure that addresses how quickly exogenous glucose can be cleared from blood. Specifically, uptake of glucose from the blood by cells is regulated by insulin. Impairment of glucose tolerance (i.e, longer time to clear given amount of glucose) indicates problems ...

[Bio101] A General EMSA (Gel-shift) Protocol

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 2/5/2011, 22245 views, 3 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.24.

[Abstract] An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe ...
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