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Journal of Biological Methods

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https://www.readbyqxmd.com/read/27660801/high-contrast-enzymatic-immunohistochemistry-of-pigmented-tissues
#1
Sara M Duncan, Gail M Seigel
Historically, standard enzyme immunohistochemistry has been accomplished with brown (DAB, diaminobenzidine) substrate. This can become problematic in pigmented tissues, such as the retina, where brown pigment of retinal pigmented epithelial (RPE) cells can be easily confounded with brown substrate. Although immunofluorescence detection methods can overcome this challenge, fluorescence may fade over a period of weeks, while enzyme substrates allow for more long-lasting, archival results. In this report, we will describe a high-contrast enzyme immunohistochemistry method ideal for pigmented tissues that utilizes purple (VIP) substrate...
2016: Journal of Biological Methods
https://www.readbyqxmd.com/read/27642613/a-rapid-fluorometric-assay-for-the-s-malonyltransacylase-fabd-and-other-sulfhydryl-utilizing-enzymes
#2
Aaron M Marcella, Adam W Barb
The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist...
2016: Journal of Biological Methods
https://www.readbyqxmd.com/read/27631018/serum-proteins-are-extracted-along-with-monolayer-cells-in-plasticware-and-interfere-with-protein-analysis
#3
Xin Hong, Yuling Meng, Steven N Kalkanis
Washing and lysing monolayer cells directly from cell culture plasticware is a commonly used method for protein extraction. We found that multiple protein bands were enriched in samples with low cell numbers from the 6-well plate cultures. These proteins contributed to the overestimation of cell proteins and led to the uneven protein loading in Western blotting analysis. In Coomassie blue stained SDS-PAGE gels, the main enriched protein band is about 69 kDa and it makes up 13.6% of total protein from 10(4) U251n cells...
2016: Journal of Biological Methods
https://www.readbyqxmd.com/read/27482532/sample-preparation-for-proteomic-analysis-using-a-gelc-ms-ms-strategy
#4
Joao A Paulo
In-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS) is a cornerstone for protein identification and characterization. Here I review this versatile approach which combines classical and modern biochemistry strategies and allows for targeted and proteome-wide analyses. Starting with any protein sample, reduced and alkylated proteins are precipitated prior to fractionation by SDS-PAGE. Proteins are in-gel digested and the resulting peptides are extracted and desalted for downstream LC-MS/MS analysis...
2016: Journal of Biological Methods
https://www.readbyqxmd.com/read/27294171/inexpensive-serotype-independent-protocol-for-native-and-bioengineered-recombinant-adeno-associated-virus-purification
#5
Erik Arden, Joseph M Metzger
Recombinant adeno-associated virus (AAV) is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes and includes bioengineered viruses. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimization to isolate purified AAV...
2016: Journal of Biological Methods
https://www.readbyqxmd.com/read/26985443/application-of-linker-technique-to-trap-transiently-interacting-protein-complexes-for-structural-studies
#6
Vishnu Priyanka Reddy Chichili, Veerendra Kumar, J Sivaraman
Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length...
2016: Journal of Biological Methods
https://www.readbyqxmd.com/read/26290880/fluorescent-foci-quantitation-for-high-throughput-analysis
#7
Elena Ledesma-Fernández, Peter H Thorpe
A number of cellular proteins localize to discrete foci within cells, for example DNA repair proteins, microtubule organizing centers, P bodies or kinetochores. It is often possible to measure the fluorescence emission from tagged proteins within these foci as a surrogate for the concentration of that specific protein. We wished to develop tools that would allow quantitation of fluorescence foci intensities in high-throughput studies. As proof of principle we have examined the kinetochore, a large multi-subunit complex that is critical for the accurate segregation of chromosomes during cell division...
July 19, 2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/25839046/inducing-gene-expression-by-targeting-promoter-sequences-using-small-activating-rnas
#8
Ji Wang, Robert F Place, Victoria Portnoy, Vera Huang, Moo Rim Kang, Mika Kosaka, Maurice Kwok Chung Ho, Long-Cheng Li
Vector-based systems comprised of exogenous nucleic acid sequences remain the standard for ectopic expression of a particular gene. Such systems offer robust overexpression, but have inherent drawbacks such as the tedious process of construction, excluding sequences (e.g. introns and untranslated regions) important for gene function and potential insertional mutagenesis of host genome associated with the use of viral vectors. We and others have recently reported that short double-stranded RNAs (dsRNAs) can induce endogenous gene expression by targeting promoter sequences in a phenomenon referred to as RNA activation (RNAa) and such dsRNAs are termed small activating RNAs (saRNAs)...
March 11, 2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26366424/generation-of-axolotl-hematopoietic-chimeras
#9
David Lopez, Edward W Scott
Wound repair is an extremely complex process that requires precise coordination between various cell types including immune cells. Unfortunately, in mammals this usually results in scar formation instead of restoration of the original fully functional tissue, otherwise known as regeneration. Various animal models like frogs and salamanders are currently being studied to determine the intracellular and intercellular pathways, controlled by gene expression, that elicit cell proliferation, differentiation, and migration of cells during regenerative healing...
February 2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26949711/isolation-and-sequencing-of-active-origins-of-dna-replication-by-nascent-strand-capture-and-release-nscr
#10
Dimiter Kunnev, Amy Freeland, Maochun Qin, Jianmin Wang, Steven C Pruitt
Nascent strand capture and release (NSCR) is a method for isolation of short nascent strands to identify origins of DNA replication. The protocol provided involves isolation of total DNA, denaturation, size fractionation on a sucrose gradient, 5'-biotinylation of the appropriate size nucleic acids, binding to a streptavidin coated magnetic beads, intensive washing, and specific release of only the RNA-containing chimeric nascent strand DNA using ribonuclease I (RNase I). The method has been applied to mammalian cells derived from proliferative tissues and cell culture but could be used for any system where DNA replication is primed by a small RNA resulting in chimeric RNA-DNA molecules...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26937420/cell-based-assays-using-calcein-acetoxymethyl-ester-show-variation-in-fluorescence-with-treatment-conditions
#11
Fayth L Miles, Jill E Lynch, Robert A Sikes
The use of fluorogenic compounds in cell and molecular biology has increased in both frequency and range of applications. However, such compounds may introduce artifacts in intracellular fluorescence and cell number estimations as a consequence of interaction with exogenous stimulants, necessitating the use of adequate controls for accurate measurements and valid conclusions. Using calcein acetoxymethyl ester (AM) in combination with various exogenous cellular treatments, we report that the standard practice of direct normalization of experimental values to controls is insufficient for fluorogenic measurements...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26618185/comprehensive-analysis-of-signal-transduction-in-three-dimensional-ecm-based-tumor-cell-cultures
#12
Iris Eke, Stephanie Hehlgans, Yaping Zong, Nils Cordes
Analysis of signal transduction and protein phosphorylation is fundamental to understanding physiological and pathological cell behavior and identifying novel therapeutic targets. Despite the fact that the use of physiological three-dimensional cell culture assays is increasing, 3D proteomics and phosphoproteomics remain challenging due to difficulties with easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates that does not compromise cell adhesion before cell lysis...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26550579/ocular-surgical-models-for-immune-and-angiogenic-responses
#13
Takenori Inomata, Alireza Mashaghi, Antonio Di Zazzo, Reza Dana
Corneal transplantation serves as a reproducible and simple surgical model to study mechanisms regulating immunity and angiogenesis. The simplicity of the model allows for systematic analysis of different mechanisms involved in immune and angiogenic privilege and their failures. This protocol describes how to induce neovessels and inflammation in an actively regulated avascular and immune-privileged site. This involves placing intra-stromal corneal sutures for two weeks, disrupting the privileges, and performing corneal transplantation subsequently...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26504887/deciphering-hepatocellular-responses-to-metabolic-and-oncogenic-stress
#14
Kathrina L Marcelo, Fumin Lin, Kimal Rajapakshe, Adam Dean, Naomi Gonzales, Cristian Coarfa, Anthony R Means, Lauren C Goldie, Brian York
Each cell type responds uniquely to stress and fractionally contributes to global and tissue-specific stress responses. Hepatocytes, liver macrophages (MΦ), and sinusoidal endothelial cells (SEC) play functionally important and interdependent roles in adaptive processes such as obesity and tumor growth. Although these cell types demonstrate significant phenotypic and functional heterogeneity, their distinctions enabling disease-specific responses remain understudied. We developed a strategy for the simultaneous isolation and quantification of these liver cell types based on antigenic cell surface marker expression...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26457320/open-source-and-diy-hardware-for-dna-nanotechnology-labs
#15
Tulsi R Damase, Daniel Stephens, Adam Spencer, Peter B Allen
A set of instruments and specialized equipment is necessary to equip a laboratory to work with DNA. Reducing the barrier to entry for DNA manipulation should enable and encourage new labs to enter the field. We present three examples of open source/DIY technology with significantly reduced costs relative to commercial equipment. This includes a gel scanner, a horizontal PAGE gel mold, and a homogenizer for generating DNA-coated particles. The overall cost savings obtained by using open source/DIY equipment was between 50 and 90%...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26457319/reliable-and-inexpensive-expression-of-large-tagged-exogenous-proteins-in-murine-bone-marrow-derived-macrophages-using-a-second-generation-lentiviral-system
#16
Matthew R Miller, Scott D Blystone
Over the past two decades, researchers have struggled to efficiently express foreign DNA in primary macrophages, impeding research progress. The applications of lipofection, electroporation, microinjection, and viral-mediated transfer typically result in disruptions in macrophage differentiation and function, low expression levels of exogenous proteins, limited efficiency and high cell mortality. In this report, after extensive optimization, we present a method of expressing large tagged proteins at high efficiency, consistency, and low cost using lentiviral infection...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26280010/a-quantitative-surface-plasmon-resonance-based-approach-to-evaluating-dna-binding-by-the-c-myc-oncoprotein-and-its-disruption-by-small-molecule-inhibitors
#17
Huabo Wang, Anand Ramakrishnan, Steven Fletcher, Edward V Prochownik
The use of small molecules to interfere with protein-protein interactions has tremendous therapeutic appeal and is an area of intense interest. Numerous techniques exist to assess these interactions and their disruption. Many, however, require large amounts of protein, do not allow interactions to be followed in real time, are technically demanding or require large capital expenditures and high levels of expertise. Surface plasmon resonance (SPR) represents a convenient alternative to these techniques with virtually none of their disadvantages...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/26146646/in-vitro-and-in-vivo-model-systems-used-in-prostate-cancer-research
#18
David Cunningham, Zongbing You
New incidence of prostate cancer is a major public health issue in the Western world, and has been rising in other areas of the globe in recent years. In an effort to understanding the molecular pathogenesis of this disease, numerous cell models have been developed, arising mostly from patient biopsies. The introduction of the genetically engineered mouse in biomedical research has allowed the development of murine models that allow for the investigation of tumorigenic and metastatic processes. Current challenges to the field include lack of an animal model that faithfully recapitulates bone metastasis of prostate cancer...
2015: Journal of Biological Methods
https://www.readbyqxmd.com/read/25606571/advanced-methods-of-microscope-control-using-%C3%AE-manager-software
#19
Arthur D Edelstein, Mark A Tsuchida, Nenad Amodaj, Henry Pinkard, Ronald D Vale, Nico Stuurman
μManager is an open-source, cross-platform desktop application, to control a wide variety of motorized microscopes, scientific cameras, stages, illuminators, and other microscope accessories. Since its inception in 2005, μManager has grown to support a wide range of microscopy hardware and is now used by thousands of researchers around the world. The application provides a mature graphical user interface and offers open programming interfaces to facilitate plugins and scripts. Here, we present a guide to using some of the recently added advanced μManager features, including hardware synchronization, simultaneous use of multiple cameras, projection of patterned light onto a specimen, live slide mapping, imaging with multi-well plates, particle localization and tracking, and high-speed imaging...
2014: Journal of Biological Methods
https://www.readbyqxmd.com/read/25530979/a-stepwise-procedure-to-test-contractility-and-susceptibility-to-injury-for-the-rodent-quadriceps-muscle
#20
Stephen J P Pratt, Richard M Lovering
In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers can also be used in measuring muscle injury, such as increased creatine kinase levels in the blood, but these are not always correlated with loss in muscle function (i.e. loss of force production). This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for loss of function...
2014: Journal of Biological Methods
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