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Mass Spectrometry-based in vitro Assay to Identify Drugs that Influence Cystine Solubility

Featured protocol,  Authors: Neelanjan Bose
Neelanjan BoseAffiliation 1: Department of Urology, University of California, San Francisco, CA, USA
Affiliation 2: Buck Institute for Research on Aging, Novato, CA, USA
For correspondence: neelanjan.bose@ucsf.edu
Bio-protocol author page: a3095
Tiffany Zee
Tiffany ZeeAffiliation 1: Department of Urology, University of California, San Francisco, CA, USA
Affiliation 2: Buck Institute for Research on Aging, Novato, CA, USA
Bio-protocol author page: a4882
Pankaj Kapahi
Pankaj KapahiAffiliation 1: Department of Urology, University of California, San Francisco, CA, USA
Affiliation 2: Buck Institute for Research on Aging, Novato, CA, USA
Bio-protocol author page: a4883
 and Marshall L. Stoller
Marshall L. StollerAffiliation: Department of Urology, University of California, San Francisco, CA, USA
For correspondence: marshal.stoller@ucsf.edu
Bio-protocol author page: a4884
date: 7/20/2017, 10 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2417.

Brief version appeared in Nat Med, Mar 2017
Cystinuria is a rare genetic disorder characterized by recurrent, painful kidney stones, primarily composed of cystine, the dimer of the amino acid cysteine (Sumorok and Goldfarb, 2013). Using a mouse model of cystinuria, we have recently shown that administration of drugs that increase cystine solubility in the urine can be a novel therapeutic strategy for the clinical management of the disease (Zee et al., 2017). There is a large unmet need in the field for developing new drugs for cystinuria. To that end, here we describe a simple in vitro cystine solubility assay that is amenable for screening compounds to identify potential drugs that may influence cystine solubility. The assay includes preparing a supersaturated solution of cystine, incubating this solution with drug(s) of choice, and finally using high pressure liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) to quantify the amount of cystine precipitated under various conditions.

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila

Featured protocol,  Authors: Ines Erdmann
Ines ErdmannAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4841
Kathrin Marter
Kathrin MarterAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
Bio-protocol author page: a4842
Oliver Kobler
Oliver KoblerAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4843
Sven Niehues
Sven NiehuesAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4844
Julia Bussmann
Julia BussmannAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4845
Anke Müller
Anke MüllerAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Bio-protocol author page: a4846
Julia Abele
Julia AbeleAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4847
Erik Storkebaum
Erik StorkebaumAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4848
Ulrich Thomas
Ulrich ThomasAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
For correspondence: thomas@lin-magdeburg.de
Bio-protocol author page: a4849
 and Daniela C. Dieterich
Daniela C. DieterichAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
For correspondence: daniela.dieterich@med.ovgu.de
Bio-protocol author page: a4850
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2397.

Brief version appeared in Nat Commun, Jul 2015
Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRSL262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

In vitro AMPylation Assays Using Purified, Recombinant Proteins

Featured protocol,  Authors: Matthias C. Truttmann
Matthias C. TruttmannAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: matthias.truttmann@childrens.harvard.edu
Bio-protocol author page: a4924
 and Hidde L. Ploegh
Hidde L. PloeghAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: hidde.ploegh@childrens.harvard.edu
Bio-protocol author page: a1563
date: 7/20/2017, 8 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2416.

Brief version appeared in Proc Natl Acad Sci U S A, Jan 2017
Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation–which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine–has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation (fic) domain-containing AMPylases sparked new interest in this PTM (Kinch et al., 2009; Yarbrough et al., 2009). Based on recent in vivo and in vitro studies, we now know that secreted bacterial AMPylases covalently attach AMP to members of the Rho family of GTPases, while metazoan AMPylases modify HSP70 family proteins in the cytoplasm and the endoplasmic reticulum (ER) (Itzen et al., 2011; Hedberg and Itzen, 2015; Truttmann and Ploegh, 2017). AMPylation is thought to trap HSP70 in a primed yet transiently disabled state that cannot participate in protein refolding reactions (Preissler et al., 2015). In vitro AMPylation experiments are key to assess the activity, kinetics and specificity of protein AMPylation catalyzed by pro- and eukaryotic enzymes. These simple assays require recombinant AMPylases, target proteins (Rho GTPases, HSP70s), as well as ATP as a nucleotide source. Here, we describe strategies to qualitatively and quantitatively study protein AMPylation in vitro.

Non-radioactive LATS in vitro Kinase Assay

Featured protocol,  Authors: Audrey W. Hong
Audrey W. HongAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
Bio-protocol author page: a4827
 and Kun-Liang Guan
Kun-Liang GuanAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
For correspondence: kuguan@ucsd.edu
Bio-protocol author page: a868
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2391.

Brief version appeared in EMBO Rep, Jan 2017
This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate.

Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC

Featured protocol,  Authors: Sheo Shankar Pandey
Sheo Shankar PandeyAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4903
Prashantee Singh
Prashantee SinghAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4904
Biswajit Samal
Biswajit SamalAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4905
Raj Kumar Verma
Raj Kumar VermaAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4906
 and Subhadeep Chatterjee
Subhadeep ChatterjeeAffiliation: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
For correspondence: subhadeep@cdfd.org.in
Bio-protocol author page: a4907
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2410.

Brief version appeared in PLoS Pathog, Nov 2016
Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology

Featured protocol,  Authors: Ran Wang
Ran WangAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
Bio-protocol author page: a4833
 and Sumaira Z. Hasnain
Sumaira Z. HasnainAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
For correspondence: sumaira.hasnain@mater.uq.edu.au
Bio-protocol author page: a4834
date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2394.

Brief version appeared in PLoS Pathog, Feb 2017
Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional roles including determining the hydration and viscoelastic properties of the mucus gel that lines and protects the intestinal epithelium. In this protocol, we comprehensively describe the method for extraction of murine mucus and its analysis by agarose gel electrophoresis. Additionally we describe the use of High Iron Diamine-Alcian Blue, Periodic Acid Schiff’s-Alcian Blue and immune–staining methods to identify and differentiate between the different states of glycosylation on these mucin glycoproteins, in particular with a focus on sulphation and sialylation.

Rice Lamina Joint Inclination Assay

Featured protocol,  Authors: Hsing-Yi Li*
Hsing-Yi LiAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4901
Hsin-Mei Wang*
Hsin-Mei WangAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4902
 and Seonghoe Jang
Seonghoe JangAffiliation 1: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Affiliation 2: Institute of Tropical Plant Science, National Cheng Kung University, Tainan, Taiwan
For correspondence: florigen@gate.sinica.edu.tw
Bio-protocol author page: a4900
 (*contributed equally to this work) date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2409.

Brief version appeared in Plant Physiol, Jan 2017
Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina joint inclination bioassays, we applied the protocol presented below. The protocol focuses on: (1) how to prepare rice samples for the assay, and (2) how to treat BL exogenously. Finally, we have added a result showing lamina inclination between WT and osbul1 in BL solutions of various concentrations.

Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions

Featured protocol,  Authors: C. Cheng Kao
C. Cheng KaoAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
For correspondence: ckao@indiana.edu
Bio-protocol author page: a4851
Ella Chuang
Ella ChuangAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4852
James Ford
James FordAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4853
Jie Huang
Jie HuangAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4854
Ram Podicheti
Ram PodichetiAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4855
 and Doug B. Rusch
Doug B. RuschAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4856
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2398.

Brief version appeared in J Virol, Aug 2016
We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.

[2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides

Featured protocol,  Authors: Marina Shenkman
Marina ShenkmanAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Bio-protocol author page: a4830
Navit Ogen-Shtern
Navit Ogen-ShternAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Present address: the Skin Research Institute, The Dead Sea and Arava Science Center, Masada, Israel
Bio-protocol author page: a4831
 and Gerardo Z. Lederkremer
Gerardo Z. LederkremerAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
For correspondence: Gerardo@post.tau.ac.il
Bio-protocol author page: a4832
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2393.

Brief version appeared in J Mol Biol, Aug 2016
Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection in a scintillation counter.

Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803

Featured protocol,  Authors: Yvonne Zilliges
Yvonne ZilligesAffiliation: Freie Universität Berlin, Institute of Experimental Physics/Biophysics and Photosynthesis, Arnimallee 14, Berlin, Germany
For correspondence: yvonne.zilliges@fu-berlin.de
Bio-protocol author page: a4874
 and Ramon Damrow
Ramon DamrowAffiliation: Humboldt-Universität zu Berlin, Institute of Biology/Biochemistry of Plants, Chausseestr. 117, Berlin, Germany
Bio-protocol author page: a4875
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2402.

Brief version appeared in Front Microbiol, Jun 2016
Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera also PHB are massively accumulated in the progression of the general nitrogen stress response. Several different technologies have been established for in situ and in vitro PHB analysis from different microbial sources. In this protocol, a rapid and reliable spectrophotometric method is described for PHB quantification in the cyanobacterium Synechocystis sp. PCC 6803 upon nitrogen deprivation as described in (Damrow et al., 2016).

The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures

Featured protocol,  Authors: Patrick Rühl
Patrick RühlAffiliation: Sulfur Biochemistry and Microbial Bioenergetics, Dept. of Biology, Technische Universität Darmstadt, Darmstadt, Germany
Bio-protocol author page: a4876
 and Arnulf Kletzin
Arnulf KletzinAffiliation: Sulfur Biochemistry and Microbial Bioenergetics, Dept. of Biology, Technische Universität Darmstadt, Darmstadt, Germany
For correspondence: kletzin@bio.tu-darmstadt.de
Bio-protocol author page: a4877
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2403.

Brief version appeared in J Bacteriol, Jan 2017
The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S0), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not required. Usually, the SOR assay is performed in a milliliter scale in S0-containing Tris-buffer at high temperatures followed by colorimetric product quantification. In order to make the SOR assay more sensitive and better reproducible, several modifications were implemented compared to the original SOR assay (Kletzin, 1989). Here we present the modified SOR assay and the following quantification of the reaction products.

Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis

Featured protocol,  Authors: Francisco J Corpas
Francisco J CorpasAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
For correspondence: javier.corpas@eez.csic.es
Bio-protocol author page: a4857
Larisse de Freitas-Silva
Larisse de Freitas-SilvaAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4864
Nuria García-Carbonero
Nuria García-CarboneroAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4865
Alba Contreras
Alba ContrerasAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4866
Fátima Terán
Fátima TeránAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4867
Carmelo Ruíz-Torres
Carmelo Ruíz-TorresAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4868
 and José M. Palma
José M. PalmaAffiliation: Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, Spanish National Research Council (CSIC), C/Profesor Albareda, 1, 18008 Granada, Spain
Bio-protocol author page: a4869
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2399.

Brief version appeared in Physiol Plant, Feb 2009
6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, chloroplast, mitochondria and peroxisomes (Corpas et al., 1998; Krepinsky et al., 2001; Mateos et al., 2009; Fernández-Fernández and Corpas, 2016; Hölscher et al., 2016). Using Arabidopsis thaliana as plant model and sweet pepper (Capsicum annuum L.) fruits as a plant with agronomical interest, this protocol illustrates how to prepare the plant extracts for the separation of the potential 6PGDH isoforms by electrophoresis on 6% polyacrylamide non-denaturing gels. Thus, this method allows detecting three 6PGDH isoforms in Arabidopsis seedlings and two 6PGDH isoforms in sweet pepper fruits.

Endpoint or Kinetic Measurement of Hydrogen Sulfide Production Capacity in Tissue Extracts

Featured protocol,  Authors: Christopher Hine
Christopher HineAffiliation: Department of Cellular and Molecular Medicine, Cleveland Clinic Lerner Research Institute, Cleveland, OH 44195 USA
For correspondence: hinec@ccf.org
Bio-protocol author page: a4788
 and James R. Mitchell
James R. MitchellAffiliation: Department of Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02115 USA
For correspondence: jmitchel@hsph.harvard.edu
Bio-protocol author page: a4789
date: 7/5/2017, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2382.

Brief version appeared in Cell, Jan 2015
Hydrogen sulfide (H2S) gas is produced in cells and tissues via various enzymatic processes. H2S is an important signaling molecule in numerous biological processes, and deficiencies in endogenous H2S production are linked to cardiovascular and other health complications. Quantitation of steady-state H2S levels is challenging due to volatility of the gas and the need for specialized equipment. However, the capacity of an organ or tissue extract to produce H2S under optimized reaction conditions can be measured by a number of current assays that vary in sensitivity, specificity and throughput capacity. We developed a rapid, inexpensive, specific and relatively high-throughput method for quantitative detection of H2S production capacity from biological tissues. H2S released into the head space above a biological sample reacts with lead acetate to form lead sulfide, which is measured on a continuous basis using a plate reader or as an endpoint assay.

Assaying Glycogen and Trehalose in Yeast

Featured protocol,  Authors: Yuping Chen
Yuping ChenAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
Bio-protocol author page: a4763
 and Bruce Futcher
Bruce FutcherAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
For correspondence: bfutcher@gmail.com
Bio-protocol author page: a4764
date: 7/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2371.

Brief version appeared in Mol Cell, May 2016
Organisms store carbohydrates in several forms. In yeast, carbohydrates are stored in glycogen (a multi-branched polysaccharide) and in trehalose (a disaccharide). As in other organisms, the amount of stored carbohydrate varies dramatically with physiological state, and accordingly, an assay of stored carbohydrate can help reveal physiological state. Here, we describe relatively easy and streamlined assays for glycogen and trehalose in yeast that can be applied either to a few samples, or in a moderately high-throughput fashion (dozens to hundreds of samples).

Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen

Featured protocol,  Authors: Brent Veerman
Brent VeermanAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a4752
 and Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: ritu.chakravarti@utoledo.edu
Bio-protocol author page: a3737
date: 7/5/2017, 189 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2367.

Brief version appeared in Arthritis Rheumatol, Jul 2015
Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases (Wang et al., 2015). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to detect the presence of autoantibodies as well as to measure the circulating autoantibodies in the sera of patients suffering with large vessel vasculitis (LVV), an autoimmune disease (Chakravarti et al., 2015). Using this assay, we detected the increase in anti-aortic antibodies in LVV patient’s sera. We have further verified the results by independent biochemical techniques and found the specificity to be > 94% (Chakravarti et al., 2015). This method can be uniquely modified to suit any autoimmune, in particular organ specific, disease and thus has wider applications in the detection and quantification of autoantibodies.

Active Cdk5 Immunoprecipitation and Kinase Assay

Featured protocol,  Authors: Andrew N. Bankston
Andrew N. BankstonAffiliation 1: Department of Pharmacology, Emory University, Atlanta, GA
Affiliation 2: Department of Neurological Surgery, University of Louisville, Louisville, KY
Bio-protocol author page: a4746
Li Ku
Li KuAffiliation: Department of Pharmacology, Emory University, Atlanta, GA
Bio-protocol author page: a4747
 and Yue Feng
Yue FengAffiliation: Department of Pharmacology, Emory University, Atlanta, GA
For correspondence: yfeng@emory.edu
Bio-protocol author page: a4748
date: 7/5/2017, 193 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2363.

Brief version appeared in J Biol Chem, Jun 2013
Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal amounts of total protein between experimental groups. Washes are then performed to remove extraneous proteins and equilibrate the Cdk5-activator complexes in the kinase buffer. Cdk5 is then incubated with histone H1, a well-established in vitro target of Cdk5, and [γ-32P]ATP. Reactions are resolved by SDS-PAGE and transferred to membranes for visualization of H1 phosphorylation and immunoblot of immunoprecipitated Cdk5 levels. We have used this assay to establish p39 as the primary activator for Cdk5 in the oligodendroglial lineage. However, this assay is amenable to other cell lineages or tissues with appropriate adjustments made to lysis conditions.

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

Featured protocol,  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, 225 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2374.

Brief version appeared in Anal Biochem, Dec 2016
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 weak non-covalent association with the tissue culture plastic. Here we describe a simple protocol for a controlled fixation of fibronectin, vitronectin and collagen IV layers by formaldehyde, which substantially enhances the stimulation of primary cell proliferation by these extracellular proteins.

Mass Spectrometry-based in vitro Assay to Identify Drugs that Influence Cystine Solubility

Authors: Neelanjan Bose
Neelanjan BoseAffiliation 1: Department of Urology, University of California, San Francisco, CA, USA
Affiliation 2: Buck Institute for Research on Aging, Novato, CA, USA
For correspondence: neelanjan.bose@ucsf.edu
Bio-protocol author page: a3095
Tiffany Zee
Tiffany ZeeAffiliation 1: Department of Urology, University of California, San Francisco, CA, USA
Affiliation 2: Buck Institute for Research on Aging, Novato, CA, USA
Bio-protocol author page: a4882
Pankaj Kapahi
Pankaj KapahiAffiliation 1: Department of Urology, University of California, San Francisco, CA, USA
Affiliation 2: Buck Institute for Research on Aging, Novato, CA, USA
Bio-protocol author page: a4883
 and Marshall L. Stoller
Marshall L. StollerAffiliation: Department of Urology, University of California, San Francisco, CA, USA
For correspondence: marshal.stoller@ucsf.edu
Bio-protocol author page: a4884
date: 7/20/2017, 10 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2417.

[Abstract] Cystinuria is a rare genetic disorder characterized by recurrent, painful kidney stones, primarily composed of cystine, the dimer of the amino acid cysteine (Sumorok and Goldfarb, 2013). Using a mouse model of cystinuria, we have recently shown that administration of drugs that increase cystine solubility in the urine can be a novel therapeutic strategy ...

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila

Authors: Ines Erdmann
Ines ErdmannAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4841
Kathrin Marter
Kathrin MarterAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
Bio-protocol author page: a4842
Oliver Kobler
Oliver KoblerAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4843
Sven Niehues
Sven NiehuesAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4844
Julia Bussmann
Julia BussmannAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4845
Anke Müller
Anke MüllerAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Bio-protocol author page: a4846
Julia Abele
Julia AbeleAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Bio-protocol author page: a4847
Erik Storkebaum
Erik StorkebaumAffiliation 1: Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster, Germany
Affiliation 2: Faculty of Medicine, University of Münster, Münster, Germany
Bio-protocol author page: a4848
Ulrich Thomas
Ulrich ThomasAffiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg, Germany
For correspondence: thomas@lin-magdeburg.de
Bio-protocol author page: a4849
 and Daniela C. Dieterich
Daniela C. DieterichAffiliation 1: Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
Affiliation 2: Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany
Affiliation 3: Center for Behavioral Brain Sciences, Magdeburg, Germany
For correspondence: daniela.dieterich@med.ovgu.de
Bio-protocol author page: a4850
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2397.

[Abstract] Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded ...

In vitro AMPylation Assays Using Purified, Recombinant Proteins

Authors: Matthias C. Truttmann
Matthias C. TruttmannAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: matthias.truttmann@childrens.harvard.edu
Bio-protocol author page: a4924
 and Hidde L. Ploegh
Hidde L. PloeghAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: hidde.ploegh@childrens.harvard.edu
Bio-protocol author page: a1563
date: 7/20/2017, 8 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2416.

[Abstract] Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation–which ...

Non-radioactive LATS in vitro Kinase Assay

Authors: Audrey W. Hong
Audrey W. HongAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
Bio-protocol author page: a4827
 and Kun-Liang Guan
Kun-Liang GuanAffiliation: Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, California 92093, USA
For correspondence: kuguan@ucsd.edu
Bio-protocol author page: a868
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2391.

[Abstract] This protocol describes a method to directly measure LATS activity by an in vitro kinase assay using YAP as a substrate....

Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC

Authors: Sheo Shankar Pandey
Sheo Shankar PandeyAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4903
Prashantee Singh
Prashantee SinghAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4904
Biswajit Samal
Biswajit SamalAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4905
Raj Kumar Verma
Raj Kumar VermaAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4906
 and Subhadeep Chatterjee
Subhadeep ChatterjeeAffiliation: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
For correspondence: subhadeep@cdfd.org.in
Bio-protocol author page: a4907
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2410.

[Abstract] Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces ...

Analyzing the Properties of Murine Intestinal Mucins by Electrophoresis and Histology

Authors: Ran Wang
Ran WangAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
Bio-protocol author page: a4833
 and Sumaira Z. Hasnain
Sumaira Z. HasnainAffiliation: Inflammatory Disease Biology and Therapeutics Group, Mater Research Institute–The University of Queensland, Translational Research Institute, Brisbane, Australia
For correspondence: sumaira.hasnain@mater.uq.edu.au
Bio-protocol author page: a4834
date: 7/20/2017, 20 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2394.

[Abstract] Specialized secretory cells known as goblet cells in the intestine and respiratory epithelium are responsible for the secretion of mucins. Mucins are large heavily glycosylated proteins and typically have a molecular mass higher than 106 Da. These large proteins are densely substituted with short glycan chains, which have many important functional ...

Rice Lamina Joint Inclination Assay

Authors: Hsing-Yi Li*
Hsing-Yi LiAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4901
Hsin-Mei Wang*
Hsin-Mei WangAffiliation: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Bio-protocol author page: a4902
 and Seonghoe Jang
Seonghoe JangAffiliation 1: Biotechnology Center in Southern Taiwan (BCST) of Agricultural Biotechnology Research Center (ABRC), Academia Sinica, Tainan, Taiwan
Affiliation 2: Institute of Tropical Plant Science, National Cheng Kung University, Tainan, Taiwan
For correspondence: florigen@gate.sinica.edu.tw
Bio-protocol author page: a4900
 (*contributed equally to this work) date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2409.

[Abstract] Brassinosteroids (BRs) promote rice lamina inclination. Recently, we showed that OsBUL1 knockout mutant rice (osbul1) is defective in brassinosteroid signaling (Jang et al., 2017). To show that lamina joint inclination of osbul1 is less-sensitive than WT to exogenous brassinolide (BL) treatment in the lamina joint inclination bioassays, we applied ...

Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions

Authors: C. Cheng Kao
C. Cheng KaoAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
For correspondence: ckao@indiana.edu
Bio-protocol author page: a4851
Ella Chuang
Ella ChuangAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4852
James Ford
James FordAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4853
Jie Huang
Jie HuangAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4854
Ram Podicheti
Ram PodichetiAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4855
 and Doug B. Rusch
Doug B. RuschAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4856
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2398.

[Abstract] We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid....

[2-3H]Mannose-labeling and Analysis of N-linked Oligosaccharides

Authors: Marina Shenkman
Marina ShenkmanAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Bio-protocol author page: a4830
Navit Ogen-Shtern
Navit Ogen-ShternAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
Present address: the Skin Research Institute, The Dead Sea and Arava Science Center, Masada, Israel
Bio-protocol author page: a4831
 and Gerardo Z. Lederkremer
Gerardo Z. LederkremerAffiliation: Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
For correspondence: Gerardo@post.tau.ac.il
Bio-protocol author page: a4832
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2393.

[Abstract] Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-3H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection ...

Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803

Authors: Yvonne Zilliges
Yvonne ZilligesAffiliation: Freie Universität Berlin, Institute of Experimental Physics/Biophysics and Photosynthesis, Arnimallee 14, Berlin, Germany
For correspondence: yvonne.zilliges@fu-berlin.de
Bio-protocol author page: a4874
 and Ramon Damrow
Ramon DamrowAffiliation: Humboldt-Universität zu Berlin, Institute of Biology/Biochemistry of Plants, Chausseestr. 117, Berlin, Germany
Bio-protocol author page: a4875
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2402.

[Abstract] Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera also PHB are ...
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[Bio101] Bradford Protein Assay

Author: Fanglian He date: 3/20/2011, 99973 views, 6 Q&A
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, 69876 views, 10 Q&A
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 ...

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, 44030 views, 2 Q&A
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 ...

[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, 43232 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....

[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, 37874 views, 3 Q&A
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 ...

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, 34354 views, 16 Q&A
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, 33012 views, 0 Q&A
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] Coomassie Blue Staining

Author: Fanglian He date: 6/5/2011, 32914 views, 0 Q&A
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, 32159 views, 4 Q&A
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, 26371 views, 3 Q&A
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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