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Bio-protocol

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https://www.readbyqxmd.com/read/29651453/bacterial-cell-wall-precursor-phosphatase-assays-using-thin-layer-chromatography-tlc-and-high-pressure-liquid-chromatography-hplc
#1
Manuel Pazos, Christian Otten, Waldemar Vollmer
Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by 'attacking' enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius ...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644261/detection-and-analysis-of-circular-rnas-by-rt-pcr
#2
Amaresh C Panda, Myriam Gorospe
Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644260/biochemical-analysis-of-dimethyl-suberimidate-crosslinked-yeast-nucleosomes
#3
Yuichi Ichiakwa, Paul D Kaufman
Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644259/rna-cap-methyltransferase-activity-assay
#4
Jackson B Trotman, Daniel R Schoenberg
Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32 P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644258/crispr-mediated-tagging-with-bira-allows-proximity-labeling-in-toxoplasma-gondii
#5
Shaojun Long, Kevin M Brown, L David Sibley
Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii . When combined with CRISPR/Cas9 based tagging, this method provides a robust approach to defining protein networks. This approach detects interaction within intact cells, it is applicable to both soluble and insoluble components, including large proteins complexes that interact with the cytoskeleton and unique microtubule organizing center that comprises the apical complex in apicomplexan parasites...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29629394/organotypic-explants-of-the-embryonic-rodent-hippocampus-an-accessible-system-for-transgenesis
#6
Archana Iyer, Lakshmi Subramanian, Shubha Tole
This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter...
March 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29651452/barnes-maze-procedure-for-spatial-learning-and-memory-in-mice
#7
Matthew W Pitts
The Barnes maze is a dry-land based rodent behavioral paradigm for assessing spatial learning and memory that was originally developed by its namesake, Carol Barnes. It represents a well-established alternative to the more popular Morris Water maze and offers the advantage of being free from the potentially confounding influence of swimming behavior. Herein, the Barnes maze experimental setup and corresponding procedures for testing and analysis in mice are described in detail.
March 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644257/intravenous-labeling-and-analysis-of-the-content-of-thymic-perivascular-spaces
#8
Roland Ruscher, Kristin A Hogquist
Following development in the thymus, T cells are thought to exit into the periphery predominantly through perivascular spaces (PVS). This exit route is used by conventional T cells, and likely also applies to unconventional T cell subsets, such as precursors of CD8αα and TCRγδ intraepithelial lymphocytes, regulatory T cells and natural killer T cells. Additional cell types might also be found in the PVS and initiate interactions with exiting T cells. The exact content of the PVS, and the processes within, are not well studied...
March 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29623285/quantification-of-bacterial-attachment-to-tissue-sections
#9
Batya Isaacson, Tehila Hadad, Gilad Bachrach, Ofer Mandelboim
Here we describe a method to test bacterial adhesion to paraffin embedded tissue sections. This method allows examining binding of different bacterial strains, transfected with a fluorescent protein reporter plasmid to various tissues, to better understand different mechanisms such as colonization. This assay provides a more physiological context to bacterial binding, than would have been achieved using adhesion assays to cell lines. The sections can be imaged using fluorescent microscopy and adhesion of various bacterial strains can be quantified and tested, simultaneously...
March 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29594188/dual-sided-voltage-sensitive-dye-imaging-of-leech-ganglia
#10
Yusuke Tomina, Daniel A Wagenaar
In this protocol, we introduce an effective method for voltage-sensitive dye (VSD) loading and imaging of leech ganglia as used in Tomina and Wagenaar (2017). Dissection and dye loading procedures are the most critical steps toward successful whole-ganglion VSD imaging. The former entails the removal of the sheath that covers neurons in the segmental ganglion of the leech, which is required for successful dye loading. The latter entails gently flowing a new generation VSD, VF2.1(OMe).H, onto both sides of the ganglion simultaneously using a pair of peristaltic pumps...
March 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29552597/synthetic-genetic-interaction-crispr-sgi-profiling-in-caenorhabditis-elegans
#11
John A Calarco, Adam D Norris
Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al ., 2005; Butland et al ., 2008; Costanzo et al ., 2010), but similar large-scale interaction studies using targeted reverse-genetic deletions in multi-cellular organisms have not been feasible. We developed a CRISPR/Cas9-based method for deleting genes in C...
March 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644256/registration-and-alignment-between-in-vivo-functional-and-cytoarchitectonic-maps-of-mouse-visual-cortex
#12
Jun Zhuang, Quanxin Wang, Marc Takeno, Jack Waters
This protocol describes a method for registration of in vivo cortical retinotopic map with cytochrome c oxidase (CO) labeled architectonic maps of the same mouse brain through the alignment of vascular fiducials. By recording surface blood vessel pattern and sequential alignment at each step, this method overcomes the challenge imposed by tissue distortion during perfusion, mounting, sectioning and histology procedures. This method can also be generalized to register and align other types of in vivo functional maps like ocular dominance map and spatial/temporal frequency tuning map with various anatomical maps of mouse cortex...
February 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644255/conditional-knockdown-of-proteins-using-auxin-inducible-degron-aid-fusions-in-toxoplasma-gondii
#13
Kevin M Brown, Shaojun Long, L David Sibley
Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii 's 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T...
February 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29600253/a-small-rna-isolation-and-sequencing-protocol-and-its-application-to-assay-crispr-rna-biogenesis-in-bacteria
#14
Sukrit Silas, Nimit Jain, Michael Stadler, Becky Xu Hua Fu, Antonio Sánchez-Amat, Andrew Z Fire, Joshua Arribere
Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, we describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis and treated with T4 polynucleotide kinase. This allows for 3' adapter ligation to CRISPR RNAs, which don't have pre-existing 3'-OH ends...
February 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29564372/adapting-the-smart-seq2-protocol-for-robust-single-worm-rna-seq
#15
Lorrayne Serra, Dennis Z Chang, Marissa Macchietto, Katherine Williams, Rabi Murad, Dihong Lu, Adler R Dillman, Ali Mortazavi
Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos , the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment...
February 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29546231/sebinger-culture-a-system-optimized-for-morphological-maturation-and-imaging-of-cultured-mouse-metanephric-primordia
#16
Mona Elhendawi, Jamie A Davies
Here, we present a detailed protocol on setting up embryonic renal organ cultures using a culture method that we have optimised for anatomical maturation and imaging. Our culture method places kidney rudiments on glass in a thin film of medium, which results in very flat cultures with all tubules in the same image plane. For reasons not yet understood, this technique results in improved renal maturation compared to traditional techniques. Typically, this protocol will result in an organ formed with distinct cortical and medullary regions as well as elongated, correctly positioned loops of Henle...
February 20, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29644254/fret-based-stoichiometry-measurements-of-protein-complexes-in-vitro
#17
Francesca Mattiroli, Yajie Gu, Karolin Luger
For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro . This assay, also known as Job plot, is set up as a continuous variation of the molar ratio between the two species, kept at constant total concentration. The FRET (Fluorescence Resonance Energy Transfer) between the two fluorescently-labeled proteins is measured and the stoichiometry is inferred from the sample with highest FRET signal...
February 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29623284/extraction-and-analysis-of-pan-metabolome-polar-metabolites-by-ultra-performance-liquid-chromatography-tandem-mass-spectrometry-uplc-ms-ms
#18
Dania M Malik, Seth Rhoades, Aalim Weljie
Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a variety of column chemistries. Recently, the comprehensive optimization of LC-MS metabolomics using design of experiments (COLMeD) approach has been described and used by our group to develop robust LC-MS workflows (Rhoades and Weljie, 2016)...
February 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29564371/tracking-endocytosis-and-intracellular-trafficking-of-epitope-tagged-syntaxin-3-by-antibody-feeding-in-live-polarized-mdck-cells
#19
Adrian J Giovannone, Elena Reales, Pallavi Bhattaram, Alberto Fraile-Ramos, Thomas Weimbs
The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes...
February 5, 2018: Bio-protocol
https://www.readbyqxmd.com/read/29527542/immunoprecipitation-of-tri-methylated-capped-rna
#20
Karen E Hayes, Jamie A Barr, Mingyi Xie, Joan A Steitz, Ivan Martinez
Cellular quiescence (also known as G0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27Kip1 . Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence...
February 5, 2018: Bio-protocol
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