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In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs

Featured protocol,  Authors: Sajni Josson
Sajni JossonAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Neostrata Inc, Princeton, USA
For correspondence: sajnij@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2138.

Brief version appeared in Oncogene, May 2015
miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of in situ hybridization (ISH) and quantum dots (QD). The advantage of using quantum dots is that several miRNA can be simultaneously measured using multiplexed QD. Additionally, miRNA can be visualized in different regions of the tissue. Since miRNA are biomarkers of various disease states, miRNA can be visualized and quantitated in tissue sections for diagnostic and prognostic purposes. Here we describe ISH-QD analysis of tissue sections. Tissue sections from xenografts or clinical specimens are used. These are deparaffinized, treated with Proteinase K and hybridized with a biotin-probe to specific to the miRNA. The in situ hybridization is performed by labeling the biotin-probes and followed by labeling with streptavidin tagged quantum dots. Image acquisition of the quantum dots is performed and analyzed for the miRNA expression levels. Combining ISH and QD gives a powerful tool to detect miRNA in different cells of the tissue.

miRNA Characterization from the Extracellular Vesicles

Featured protocol,  Authors: Sajni Josson
Sajni JossonAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Neostrata Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 33 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2139.

Brief version appeared in Oncogene, May 2015
Cancer cells and cancer associated stromal cells co-evolve secrete extracelluar vesicles to the surrounding regions and regulate several processes involved in cancer metastasis. miRNAs have been known to be mediators of cancer progression and metastasis. miRNAs consist of short noncoding RNA. miRNAs are stable in extracellular fluids such as serum, plasma and urine. miRNAs are secreted by cells in normal and diseased conditions. miRNAs signatures have been identified specific to certain disease conditions. Therefore they are valuable biomarkers for different diseases. In our study we identified certain miRNAs, miR-409-3p and miR-409-5p, which were secreted by activated stromal fibroblast cells and were taken up by cancer cells to induce explosive tumor growth, through activation of epithelial to mesenchymal transition of cancer cells. Here we describe a procedure to determine miRNAs (miR-409-3p and miR-409-5p) in extracellular vesicles, which were secreted by prostate cancer stromal cells expressing miR-409. In this procedure, conditioned media from the stromal fibroblasts was used to extract the vesicular fraction. RNA was purified from the vesicular fraction, and specific miRNA was reverse transcribed and quantitated using real-time PCR assay.

Rice Root Organic Acid Efflux Measurement by Using Ion Chromatography

Featured protocol,  Authors: Chun-quan Zhu
Chun-quan ZhuAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4131
Xiao-fang Zhu
Xiao-fang Zhu Affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4132
 and Ren-fang Shen
Ren-fang ShenAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
For correspondence: rfshen@issas.ac.cn
Bio-protocol author page: a4133
date: 2/20/2017, 52 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2141.

Brief version appeared in Plant Physiol, Jun 2016
Organic acids secreted from plant roots play important roles in various biological processes including nutrient acquisition, metal detoxification, and pathogen attraction. The secretion of organic acids may be affected by various conditions such as plant growth stage, nutrient deficiency, and abiotic stress. For example, when white lupin (Lupinus albus L.) is exposed to phosphorus (P)-deficient conditions, the secretion of citrate acid from its proteoid roots significantly increases (Neumann et al., 1999). This protocol describes a method for the collection and measurement of the efflux of organic acids (oxalate, malate, and citrate) from the roots of rice cultivar Nipponbare (‘Nip’) under different nitrogen forms (NH4+ and NO3-), together with different P supply (+P and -P) conditions.

Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity

Featured protocol,  Authors: Qiang Chen
Qiang ChenAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
Bio-protocol author page: a4118
Lingling Yu
Lingling YuAffiliation: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: 94116758@qq.com
Bio-protocol author page: a4119
Xiao Tan
Xiao Tan Affiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: xiao-tan@hotmail.com
Bio-protocol author page: a4120
 and Sen Liu
Sen LiuAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: senliu.ctgu@gmail.com
Bio-protocol author page: a4117
date: 2/20/2017, 39 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2140.

Brief version appeared in Sci Rep, Apr 2016
Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria are the simplest and one of the best studied models. They contain the three clock proteins KaiA, KaiB, and KaiC which can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol.

Chromatographic Separation of the Codonocarpine Type Alkaloids from the Root Bark of Capparis decidua

Featured protocol,  Authors: Yvonne Forster
Yvonne ForsterAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4136
Abdul Ghaffar
Abdul GhaffarAffiliation: Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
Bio-protocol author page: a4135
 and Stefan Bienz
Stefan BienzAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
For correspondence: stefan.bienz@chem.uzh.ch
Bio-protocol author page: a4137
date: 2/20/2017, 41 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2144.

Brief version appeared in Phytochemistry, Aug 2016
Various parts of the caper tree Capparis decidua have found application in traditional medicine. The isolation and structural elucidation of the codonocarpine type alkaloids contained in the root bark, however, is not trivial and has probably led to misinterpretation in the past. This protocol describes the extraction and chromatographic separation of the four major alkaloids of the root bark of Capparis decidua. The delivered samples of cadabicine, codonocarpine, isocodonocarpine and capparidisinine were suitable for their unambiguous structural elucidation by NMR, MS and MS/MS.

Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies

Featured protocol,  Authors: Yung-Ning Chang
Yung-Ning ChangAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
Bio-protocol author page: a4040
Farooque R. Shaik
Farooque R. ShaikAffiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
Bio-protocol author page: a4041
Yvonne Neldner
Yvonne NeldnerAffiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4042
 and Eric R. Geertsma
Eric R. GeertsmaAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
For correspondence: geertsma@em.uni-frankfurt.de
Bio-protocol author page: a4043
date: 2/5/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2116.

Brief version appeared in Nat Struct Mol Biol, Oct 2015
The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, termed SLC26Dg, from Deinococcus geothermalis. Following these instructions we obtained well-folded and homogenous material of the membrane protein SLC26Dg and the nanobody Nb5776 that enabled us to crystallize the complex and determine its structure (Geertsma et al., 2015). The procedure may be adapted to purify and crystallize other membrane protein complexes.

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

Featured protocol,  Authors: Shengping Li
Shengping LiAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4023
Yanhe Zhao
Yanhe ZhaoAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4024
 and Yunkun Wu
Yunkun WuAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
For correspondence: wuyk@fjirsm.ac.cn
Bio-protocol author page: a4025
date: 2/5/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2122.

Brief version appeared in Plant Cell, May 2015
GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is about 350-450 resides and can be subdivided into five distinct motifs with their name derived from the most prominent amino acids: LRI (leucine-rich region I), VHIID, LRII (leucine-rich region II), PFYRE and SAW and mainly function in the interaction between GRAS proteins and their partners (Sun et al., 2012).By phylogenetic analysis, the GRAS family can be divided into more than ten subfamilies, of which SCL4/7 is one important subgroup and functions in response to environmental stresses. Here we describe a detailed protocol forthe expression and purification of the GRAS domain of Os-SCL7, a SCL4/7 member in rice, which enable us to crystallize it and determine its structure.

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

Featured protocol,  Authors: Anna Vangone
Anna VangoneAffiliation: Computational Structural Biology group, Bijvoet Center for Biomolecular Research, Faculty of Science Chemistry, Utrecht University, Utrecht, the Netherlands
For correspondence: a.vangone@uu.nl
Bio-protocol author page: a4057
 and Alexandre M. J. J. Bonvin
Alexandre M. J. J. BonvinAffiliation: Computational Structural Biology group, Bijvoet Center for Biomolecular Research, Faculty of Science Chemistry, Utrecht University, Utrecht, the Netherlands
For correspondence: a.m.j.j.bonvin@uu.nl
Bio-protocol author page: a4058
date: 2/5/2017, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2124.

Brief version appeared in eLife, Jul 2015
Biomolecular interactions between proteins regulate and control almost every biological process in the cell. Understanding these interactions is therefore a crucial step in the investigation of biological systems and in drug design. Many efforts have been devoted to unravel principles of protein-protein interactions. Recently, we introduced a simple but robust descriptor of binding affinity based only on structural properties of a protein-protein complex. In Vangone and Bonvin (2015), we demonstrated that the number of interfacial contacts at the interface of a protein-protein complex correlates with the experimental binding affinity. Our findings have led one of the best performing predictor so far reported (Pearson’s Correlation r = 0.73; RMSE = 1.89 kcal mol-1). Despite the importance of the topic, there is surprisingly only a limited number of online tools for fast and easy prediction of binding affinity. For this reason, we implemented our predictor into the user-friendly PRODIGY web-server. In this protocol, we explain the use of the PRODIGY web-server to predict the affinity of a protein-protein complex from its three-dimensional structure. The PRODIGY server is freely available at: http://milou.science.uu.nl/services/PRODIGY.

In vitro Assessment of RNA Polymerase I Activity

Featured protocol,  Author: Marzia Govoni
Marzia GovoniAffiliation: Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy
For correspondence: marzia.govoni@unibo.it
Bio-protocol author page: a4032
date: 2/5/2017, 149 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2120.

Brief version appeared in Oncogene, Feb 2016
In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the smallest species of ribosomal RNA (5S rRNA). This protocol describes an in vitro assay to evaluate the rRNA transcriptional activity of RNA polymerase I. The method measures the quantity of radiolabelled uridine 5’ triphosphate incorporated in ex novo synthesized rRNA molecules by RNA polymerase I, in optimal conditions for the enzyme activity and in the presence of a toxin, α-amanitin, which inhibits RNA polymerase II and III without affecting RNA polymerase I (Novello and Stirpe, 1970).

In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs

Authors: Sajni Josson
Sajni JossonAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Neostrata Inc, Princeton, USA
For correspondence: sajnij@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Present address: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 36 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2138.

[Abstract] miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of in situ hybridization (ISH) and quantum dots (QD). The advantage of using quantum dots is that several miRNA can be simultaneously ...

miRNA Characterization from the Extracellular Vesicles

Authors: Sajni Josson
Sajni JossonAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Neostrata Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4072
Murali Gururajan
Murali GururajanAffiliation 1: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
Affiliation 2: Bristol-Myers Squibb Inc, Princeton, USA
For correspondence: gururajanmurali@gmail.com
Bio-protocol author page: a4073
 and Leland W.K. Chung
Leland W.K. ChungAffiliation: Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA
For correspondence: leland.chung@cshs.org
Bio-protocol author page: a4074
date: 2/20/2017, 33 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2139.

[Abstract] Cancer cells and cancer associated stromal cells co-evolve secrete extracelluar vesicles to the surrounding regions and regulate several processes involved in cancer metastasis. miRNAs have been known to be mediators of cancer progression and metastasis. miRNAs consist of short noncoding RNA. miRNAs are stable in extracellular fluids such as serum, ...

Rice Root Organic Acid Efflux Measurement by Using Ion Chromatography

Authors: Chun-quan Zhu
Chun-quan ZhuAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4131
Xiao-fang Zhu
Xiao-fang Zhu Affiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
Bio-protocol author page: a4132
 and Ren-fang Shen
Ren-fang ShenAffiliation: State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing, China
For correspondence: rfshen@issas.ac.cn
Bio-protocol author page: a4133
date: 2/20/2017, 52 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2141.

[Abstract] Organic acids secreted from plant roots play important roles in various biological processes including nutrient acquisition, metal detoxification, and pathogen attraction. The secretion of organic acids may be affected by various conditions such as plant growth stage, nutrient deficiency, and abiotic stress. For example, when white lupin (Lupinus albus ...

Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity

Authors: Qiang Chen
Qiang ChenAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
Bio-protocol author page: a4118
Lingling Yu
Lingling YuAffiliation: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: 94116758@qq.com
Bio-protocol author page: a4119
Xiao Tan
Xiao Tan Affiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: xiao-tan@hotmail.com
Bio-protocol author page: a4120
 and Sen Liu
Sen LiuAffiliation 1: Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, China
Affiliation 2: College of Medical Science, China Three Gorges University, Yichang, China
For correspondence: senliu.ctgu@gmail.com
Bio-protocol author page: a4117
date: 2/20/2017, 39 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2140.

[Abstract] Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria ...

Chromatographic Separation of the Codonocarpine Type Alkaloids from the Root Bark of Capparis decidua

Authors: Yvonne Forster
Yvonne ForsterAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4136
Abdul Ghaffar
Abdul GhaffarAffiliation: Department of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
Bio-protocol author page: a4135
 and Stefan Bienz
Stefan BienzAffiliation: Department of Chemistry, University of Zurich, Zurich, Switzerland
For correspondence: stefan.bienz@chem.uzh.ch
Bio-protocol author page: a4137
date: 2/20/2017, 41 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2144.

[Abstract] Various parts of the caper tree Capparis decidua have found application in traditional medicine. The isolation and structural elucidation of the codonocarpine type alkaloids contained in the root bark, however, is not trivial and has probably led to misinterpretation in the past. This protocol describes the extraction and chromatographic separation ...

Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies

Authors: Yung-Ning Chang
Yung-Ning ChangAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
Bio-protocol author page: a4040
Farooque R. Shaik
Farooque R. ShaikAffiliation: Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland
Bio-protocol author page: a4041
Yvonne Neldner
Yvonne NeldnerAffiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland
Bio-protocol author page: a4042
 and Eric R. Geertsma
Eric R. GeertsmaAffiliation: Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany
For correspondence: geertsma@em.uni-frankfurt.de
Bio-protocol author page: a4043
date: 2/5/2017, 138 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2116.

[Abstract] The SLC26 or SulP proteins constitute a large family of anion transporters that are ubiquitously expressed in pro- and eukaryotes. In human, SLC26 proteins perform important roles in ion homeostasis and malfunctioning of selected members is associated with diseases. This protocol details the production and crystallization of a prokaryotic SLC26 homolog, ...

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

Authors: Shengping Li
Shengping LiAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4023
Yanhe Zhao
Yanhe ZhaoAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
Bio-protocol author page: a4024
 and Yunkun Wu
Yunkun WuAffiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China
For correspondence: wuyk@fjirsm.ac.cn
Bio-protocol author page: a4025
date: 2/5/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2122.

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

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

Authors: Anna Vangone
Anna VangoneAffiliation: Computational Structural Biology group, Bijvoet Center for Biomolecular Research, Faculty of Science Chemistry, Utrecht University, Utrecht, the Netherlands
For correspondence: a.vangone@uu.nl
Bio-protocol author page: a4057
 and Alexandre M. J. J. Bonvin
Alexandre M. J. J. BonvinAffiliation: Computational Structural Biology group, Bijvoet Center for Biomolecular Research, Faculty of Science Chemistry, Utrecht University, Utrecht, the Netherlands
For correspondence: a.m.j.j.bonvin@uu.nl
Bio-protocol author page: a4058
date: 2/5/2017, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2124.

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

In vitro Assessment of RNA Polymerase I Activity

Author: Marzia Govoni
Marzia GovoniAffiliation: Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy
For correspondence: marzia.govoni@unibo.it
Bio-protocol author page: a4032
date: 2/5/2017, 149 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2120.

[Abstract] In eukaryotic cells transcriptional processes are carried out by three different RNA polymerases: RNA polymerase I which specifically transcribes ribosomal RNA (rRNA), RNA polymerase II which transcribes protein-coding genes to yield messenger RNAs (mRNAs) and small RNAs, while RNA polymerase III transcribes the genes for transfer RNAs and for the ...

Nitrate Assay for Plant Tissues

Authors: Lufei Zhao
Lufei ZhaoAffiliation: State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, China
For correspondence: lufeizh@163.com
Bio-protocol author page: a3770
 and Yong Wang
Yong WangAffiliation: State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, China
Bio-protocol author page: a3771
date: 1/20/2017, 413 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2029.

[Abstract] Nitrogen is an essential macronutrient for plant growth and nitrate content in plants can reflect the nitrogen supply of soil. Here, we provide the salicylic acid method to evaluate the nitrate content in plant tissues. The method is reliable and stable, thus it can be a good choice for measurement of nitrate in plant tissues....
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[Bio101] Bradford Protein Assay

Author: Fanglian He date: 3/20/2011, 79429 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, 61561 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 ...

[Bio101] GST-Pull Down Protocol

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

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

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, 39443 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 ...

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, 31197 views, 15 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] 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, 30805 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 ...

[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, 30176 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, 28911 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, 28789 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, 23332 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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