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RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs

Authors: Jeremy G. Bird
Jeremy G. BirdAffiliation 1: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, USA
Affiliation 2: Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, USA
Bio-protocol author page: a4666
, Bryce E. Nickels
Bryce E. NickelsAffiliation: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, USA
For correspondence: bnickels@waksman.rutgers.edu
Bio-protocol author page: a4667
 and Richard H. Ebright
Richard H. EbrightAffiliation: Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, USA
For correspondence: ebright@waksman.rutgers.edu
Bio-protocol author page: a4668
,  date: 6/20/2017, 200 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2336.

[Abstract] It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA polymerases ...

Producing GST-Cbx7 Fusion Proteins from Escherichia coli

Authors: Thao Ngoc Huynh
Thao Ngoc HuynhAffiliation: Department of Chemistry, University of Colorado Denver, Denver, CO, USA
Bio-protocol author page: a4652
 and Xiaojun Ren
Xiaojun Ren Affiliation: Department of Chemistry, University of Colorado Denver, Denver, CO, USA
For correspondence: xiaojun.ren@ucdenver.edu
Bio-protocol author page: a4653
,  date: 6/20/2017, 223 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2333.

[Abstract] This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside and they were purified ...

Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System

Authors: Reka A. Haraszti
Reka A. HarasztiAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4671
, Andrew Coles
Andrew ColesAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4672
, Neil Aronin
Neil AroninAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4673
, Anastasia Khvorova
Anastasia KhvorovaAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
Bio-protocol author page: a4674
 and Marie-Cécile Didiot
Marie-Cécile DidiotAffiliation 1: RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA
Affiliation 2: Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
For correspondence: marie.didiot@umassmed.edu
Bio-protocol author page: a4675
,  date: 6/20/2017, 206 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2338.

[Abstract] Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural ...

Expression and Purification of the Cas10-Csm Complex from Staphylococci

Authors: Lucy Chou-Zheng
Lucy Chou-ZhengAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, USA
Bio-protocol author page: a4647
 and Asma Hatoum-Aslan
Asma Hatoum-AslanAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, USA
For correspondence: asma.hatoum@ua.edu
Bio-protocol author page: a4491
,  date: 6/5/2017, 402 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2353.

[Abstract] CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a class of prokaryotic immune systems that degrade foreign nucleic acids in a sequence-specific manner. These systems rely upon ribonucleoprotein complexes composed of Cas nucleases and small CRISPR RNAs (crRNAs). Staphylococcus epidermidis and Staphylococcus ...

Induction and Quantification of Patulin Production in Penicillium Species

Authors: Yong Chen
Yong ChenAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
Bio-protocol author page: a4471
, Boqiang Li
Boqiang LiAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
Bio-protocol author page: a1847
, Zhanquan Zhang
Zhanquan ZhangAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
Bio-protocol author page: a1845
 and Shiping Tian
Shiping TianAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
For correspondence: tsp@ibcas.ac.cn
Bio-protocol author page: a1848
,  date: 6/5/2017, 268 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2324.

[Abstract] Patulin, a worldwide regulated mycotoxin, is primarily produced by Penicillium and Aspergillus species during fruit spoilage. Patulin contamination is a great concern with regard to human health because exposure of the mycotoxin can result in severe acute and chronic toxicity, including neurotoxic, mutagenic, and immunotoxic effects. Penicillium expansum ...

Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC

Authors: Chika Yasuda
Chika YasudaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4452
, Hiroko Iijima
Hiroko IijimaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4453
, Haruna Sukigara
Haruna SukigaraAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4454
 and Takashi Osanai
Takashi OsanaiAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
For correspondence: tosanai@meiji.ac.jp
Bio-protocol author page: a4455
,  date: 5/5/2017, 359 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2257.

[Abstract] Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with ...

Preparation of Everted Membrane Vesicles from Escherichia coli Cells

Author: Marina Verkhovskaya
Marina VerkhovskayaAffiliation: Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Helsinki, Finland
For correspondence: Marina.Verkhovskaya@Helsinki.Fi
Bio-protocol author page: a4446
,  date: 5/5/2017, 349 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2254.

[Abstract] The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps. ...

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

Authors: Forrest C. Walker
Forrest C. WalkerAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
Bio-protocol author page: a4490
 and Asma Hatoum-Aslan
Asma Hatoum-AslanAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
For correspondence: ahatoum@ua.edu
Bio-protocol author page: a4491
,  date: 5/5/2017, 513 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2293.

[Abstract] CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. ...

Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria

Authors: Carlos Eduardo Pouey Cunha*
Carlos Eduardo Pouey CunhaAffiliation 1: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Affiliation 2: Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil
Bio-protocol author page: a4431
, Jane Newcombe*
Jane NewcombeAffiliation: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Bio-protocol author page: a4432
, Odir Antonio Dellagostin
Odir Antonio DellagostinAffiliation: Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil
Bio-protocol author page: a4433
 and Johnjoe McFadden
Johnjoe McFaddenAffiliation: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
For correspondence: j.mcfadden@surrey.ac.uk
Bio-protocol author page: a4434
 (*contributed equally to this work),  date: 5/5/2017, 426 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2250.

[Abstract] The meningococcus (Neisseria meningitidis) remains an important threat to human health worldwide. This Gram-negative bacterium causes elevated disabilities and mortality in infected individuals. Despite several available vaccines, currently there is no universal vaccine against all circulating meningococcal strains (Vogel et al., 2013). Herein, we ...

RNA-dependent RNA Polymerase Assay for Hepatitis E Virus

Authors: Vidya P. Nair
Vidya P. NairAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
Bio-protocol author page: a4217
, Saumya Anang
Saumya AnangAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
Bio-protocol author page: a4292
, Akriti Srivastava
Akriti SrivastavaAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
Bio-protocol author page: a4293
 and Milan Surjit
Milan SurjitAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
For correspondence: milan@thsti.res.in
Bio-protocol author page: a4218
,  date: 4/5/2017, 715 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2199.

[Abstract] RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) RdRp. In contrast to protocols available to assay ...
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[Bio101] Lentivirus Production

Author: Nabila Aboulaich,  date: 3/5/2011, 24304 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.39.

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid....

In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
,  date: 6/5/2012, 22643 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

[Bio101] Purification of 6x His-tagged Protein (from E. coli)

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
,  date: 1/5/2011, 14082 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.8.

[Abstract] A polyhistidine-tag is an amino acid motif that contains at least six histidine (His) residues, usually at the N- or C-terminus of the protein. This tag can also be referred to as a hexa histidine-tag or a 6x His-tag. The protocol described here has been developed to purify His-tagged proteins from ...

[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicin, Baltimore , USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
,  date: 1/5/2011, 11502 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.12.

[Abstract] This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA....

[Bio101] Expression and Purification of GST-tagged Proteins from E. coli

Author: Lin Fang
Lin FangAffiliation: Department of Pediatrics, School of Medicine, Stanford University, Stanford, USA
For correspondence: cheerfulfang@hotmail.com
Bio-protocol author page: a20
,  date: 9/20/2011, 11286 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.132.

[Abstract] This protocol describes a method for the small and large-scale expression and purification of GST proteins. Due to the diverse nature of proteins, a small-scale expression and purification test is always recommended....

[Bio101] Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
,  date: 1/5/2011, 11023 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.15.

[Abstract] In this protocol, we show how to purify membrane proteins from yeast using affinity purification under native conditions at a small scale. ...

Co-immunoprecipitation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
,  date: 8/20/2012, 9991 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.250.

[Abstract] This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically ...

Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins

Authors: Timo A. Lehti
Timo A. LehtiAffiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland
For correspondence: timo.lehti@helsinki.fi
Bio-protocol author page: a809
 and Benita Westerlund-Wikström
Benita Westerlund-WikströmAffiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a810
,  date: 9/5/2013, 9114 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.888.

[Abstract] This simple protocol describes how to detect antigens from agar-grown bacterial colonies transferred to nitrocellulose using specific antibodies. The protocol is well suitable for detection of bacterial proteins exposed on the cell surface or secreted to the extracellular space and it can be modified ...

[Bio101] TAP Purification of Yeast Proteins

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
,  date: 1/5/2011, 8868 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.17.

[Abstract] Tandem affinity purification (TAP) is used to look at protein-protein interaction. Its use relies on generating a fusion protein with a TAP tag on the C- or N- terminal end. In this protocol, a two-step purification of N-terminus TAP-tagged proteins from yeast is described....

Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs

Authors: Jinglin L. Xie
Jinglin L. XieAffiliation: Molecular Genetics, University of Toronto, Toronto, Canada
Bio-protocol author page: a1568
, Sheena D. Singh-Babak
Sheena D. Singh-BabakAffiliation: Department of Immunology & Microbiology, University of California, San Francisco, San Francisco, USA
Bio-protocol author page: a1569
 and Leah E. Cowen
Leah E. CowenAffiliation: Molecular Genetics, University of Toronto, Toronto, Canada
For correspondence: leah.cowen@utoronto.ca
Bio-protocol author page: a295
,  date: 10/20/2012, 8337 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.252.

[Abstract] The Minimum Inhibitory Concentration (MIC) Assay is widely used to measure the susceptibility of yeasts to antifungal agents. In serial two-fold dilutions, the lowest concentration of antifungal drug that is sufficient to inhibit fungal growth is the MIC. Typically, 50% inhibitory (MIC50) or 80% inhibitory ...
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