Notes

Overexpression involving SHMT2 Predicts a Poor Prospects along with Stimulates Tumour Cellular Rise in Vesica Cancer.
Serine palmitoyltranferase (SPT) is a pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be studied in both cells and in a cell free system. This assay procedure is applicable to any type of mammalian cell. Cyclopamine Here we provide the detailed protocol to measure SPT activity in the presence of either short chain (C8-ceramide) or long chain ceramide (C24-ceramide). One of the greatest advantages of this protocol is the ability to test insoluble long chain ceramides. We accomplished this by generating long chain ceramide through endogenous ceramide synthase by providing exogenous sphingosine and 241 acyl CoA in HeLa cell membranes. This SPT assay procedure is simple and easy to perform and does not require sophisticated instruments.Human leukocyte antigen class I (HLA-I) molecules are a group of structurally-related cell surface proteins with a high degree of variability within the population. While only up to six variants are expressed in an individual person, the whole population contains thousands of different variants. The ability to distinguish specific variants is important in the clinic to determine compatibility during organ and bone marrow transplantation and in the laboratory to study the biological properties of individual variants. Solid phase bead arrays contain purified, individually identifiable HLA-I molecules that can be used to determine antibody specificity for individual HLA-I proteins. This method is high-throughput, highly specific, and allows for simultaneous screening of antibodies against multiple HLA-I allotypes. The beads are particularly useful for screening patient sera for the presence of donor-specific antibodies against individual HLA-I variants (which can arise during pregnancy, blood transfusion, or organ transplantation). Alternate approaches, such as the use of individual HLA-I-expressing cell lines, are more time consuming, and such cell lines are difficult to procure and standardize. The HLA-I beads are also useful to study HLA-I specificity and selectivity for other receptors and binding partners.Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively low-throughput such that collecting enough data to fully characterize populations of cells is challenging. Flow cytometry avoids this drawback, and is a powerful tool that allows for rapid, high-throughput measurement of fluorescent readouts in individual cells. When combined with pH-sensitive fluorescent reporters, it can be used to characterize the intracellular pH of large populations of cells at the single-cell level. We adapted microscopy and flow-cytometry based methods to measure the intracellular pH of yeast. Cells can be grown under near-native conditions up until the point of measurement, and the protocol can be adapted to single-point or dynamic (time-resolved) measurements during changing environmental conditions.The measurement of single cell size remains an obstacle towards a deeper understanding of cell growth control, tissue homeostasis, organogenesis, and a wide range of pathologies. Recent advances have placed a spotlight on the importance of cell volume in the regulation of fundamental cell signaling pathways including those known to orchestrate progression through the cell cycle. Here we provide our protocol for the Fluorescence Exclusion Method (FXm); references to the development of FXm; and a brief outlook on future advances in image analysis which may expand the range of problems studied utilizing FXm as well as lower the barrier to entry for groups interested in adding cell volume measurements into their experimental repertoire.The novel object recognition (NOR) task is a behavioral test commonly used to evaluate episodic-like declarative memory and it relies on the innate tendency of rodents to explore novelty. Here we present a maze used to evaluate NOR memory in mice that reduces the time of the assay while improving reliability of the measurements by increasing the exploratory behavior. This memory test, being performed in a two-arms maze, is suitable for several strains of mice (including inbreed and outbreed) and does not require extended training sessions allowing an accurate temporal assessment of memory formation. This particular maze increases the mouse exploration time and reduces variability compared to other arenas used before to assess NOR. As both long- and short-term NOR memory can be easily and accurately quantified using this paradigm, this improved methodology can be easily applied to study pharmacological, genetic or age-related modulation of cognitive function.The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined the orientation of Saccharalobus solfataricus (Sso) Minichromosome maintenance (MCM) helicase at a DNA fork utilizing a site-specific DNA cleavage and mapping assay. Here, we describe a detailed protocol for site-specific DNA footprinting using 4-azidophenacyl bromide (APB). This method provides a straightforward, biochemical method to reveal the DNA binding orientation of SsoMCM helicase and can be applied to other DNA binding proteins.Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological importance of post-transcriptional RNA modifications is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. By following the maturation of a model yeast tRNA with time-resolved NMR, we showed that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. The implementation of this method requires the production for NMR spectroscopy of tRNA samples with different modification status, in order to identify the NMR signature of individual modifications. The production of tRNA samples for the analysis of modification pathways with NMR spectroscopy will be presented here and examplified on the yeast tRNAPhe, but can be extended to any other tRNA by changing the sequence of the construct. The protocol describes the production of unmodified tRNA samples by in vitro transcription, and the production of modified tRNA samples by recombinant expression of tRNAs in E. coli.RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine sequencers. In order to reduce cost, we used cost-effective and robust commercial regents with small reaction volumes. This method is a whole mRNA-Seq, which can be applied even to non-model organisms lacking the transcriptome references. In addition, we designed large number of 3' PCR primer including 8 nucleotides barcode sequences for multiplexing up to three hundreds samples. To summarize, it is possible with this protocol to prepare 96 directional RNA-Seq libraries from purified total RNA in three days and can be pooled for up to three hundred libraries. This is beneficial for large scale transcriptome analysis in many fields of animals and plant biology.
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