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Journal of Biological Methods

Laura Johnson Battle, Timothy C Chambers
Protein phosphorylation and dephosphorylation reactions play key regulatory roles in many fundamental cellular processes. Due to the large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging. However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation...
2017: Journal of Biological Methods
Karyn Schmidt, Frank Buquicchio, Johanna S Carroll, Robert J Distel, Carl D Novina
Long non-coding RNAs (lncRNAs) regulate critical cellular processes and their dysregulation contributes to multiple diseases. Although only a few lncRNAs have defined mechanisms, many of these characterized lncRNAs interact with transcription factors to regulate gene expression, suggesting a common mechanism of action. Identifying RNA-bound transcription factors is especially challenging due to inefficient RNA immunoprecipitation and low abundance of many transcription factors. Here we describe a highly sensitive, user-friendly, and inexpensive technique called RATA (RNA-associated transcription factor array), which utilizes a MS2-aptamer pulldown strategy coupled with transcription factor activation arrays for identification of transcription factors associated with a nuclear RNA of interest...
2017: Journal of Biological Methods
P M Dingess, M J Deters, R A Darling, E A Yarborough, T E Brown
Drug addicts are extremely sensitive to cues that predict drug availability and exposure to these cues can facilitate drug relapse. Cues vary in their nature but can include drug-associated paraphernalia, environmental contexts, and discrete conditioned stimuli (e.g., advertisements). One cue that has recently been heavily investigated is that of social interaction. To date, it has been demonstrated that when cocaine is conditioned with social interaction, place preference for cocaine significantly increases, suggesting that the presence of social interaction during a drug-associated "high" enhances the magnitude of drug reward...
2017: Journal of Biological Methods
Mario D Escobar, Jason L Hunt
Ribonucleic acid (RNA) is widely used in molecular biology assays, and some of the most common assays include: northern blotting and RT-PCR gene expression analysis. RNA is generally extracted by two methods: phenol-chloroform or commercially available silica spin column kits. Phenol-chloroform extraction is generally more economical; however, it produces hazardous byproducts, and leftover chemicals in the sample that can inhibit downstream applications. Commercial kits usually have simple set ups and short preparation time; however, they can introduce a significant expense to laboratory budgets...
2017: Journal of Biological Methods
Natasha Govindasamy, MengJie Yan, Paul Jurasz
Gelatin zymography and immunoblot are widely used gel electrophoresis techniques to study matrix metalloproteinases-2 and -9. Each method has its advantages and disadvantages. Zymography is exquisitely sensitive but offers no loading control to ensure equal sample loading. Immunoblot is a 100-1000-fold less sensitive, but allows for the probing of a sample loading control such as β-actin to ensure accurate protein loading. In this report, we describe two simple protocols that combine gelatin zymography to study MMP-2 and -9 levels with an in-gel β-actin immunoblot loading control, thus combining sensitivity and accuracy in a single assay...
November 1, 2016: Journal of Biological Methods
Matthias Totzeck, Ulrike B Hendgen-Cotta, Brent A French, Tienush Rassaf
Although urgently needed in clinical practice, a cardioprotective therapeutic approach against myocardial ischemia/ reperfusion injury remains to be established. Remote ischemic preconditioning (rIPC) and ischemic preconditioning (IPC) represent promising tools comprising three entities: the generation of a protective signal, the transfer of the signal to the target organ, and the response to the transferred signal resulting in cardioprotection. However, in light of recent scientific advances, many controversies arise regarding the efficacy of the underlying signaling...
2016: Journal of Biological Methods
Hector A Lucero, Shenia Patterson, Shinobu Matsuura, Katya Ravid
Bone marrow (BM) reticulin fibrosis (RF), revealed by silver staining of tissue sections, is associated with myeloproliferative neoplasms, while tools for quantitative assessment of reticulin deposition throughout a femur BM are still in need. Here, we present such a method, allowing via analysis of hundreds of composite images to identify a patchy nature of RF throughout the BM during disease progression in a mouse model of myelofibrosis. To this end, initial conversion of silver stained BM color images into binary images identified two limitations: variable color, owing to polychromatic staining of reticulin fibers, and variable background in different sections of the same batch, limiting application of the color deconvolution method, and use of constant threshold, respectively...
2016: Journal of Biological Methods
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
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
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
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
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
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
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
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
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
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
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
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
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
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