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Biochemistry

Immunoprecipitation of ROR1 Authors:  Vincent T. Bicocca and Jeffrey W. Tyner, date: 09/20/2013, view: 3204, Q&A: 0
ROR1 is a receptor tyrosine kinase family member studied for its roles in development and cancer. Here we describe a protocol for immunoprecipitation of endogenous ROR1 from t(1;19) (a disease subtype categorized by its chromosome translocation) acute lymphoblastic leukemia immortalized cell lines.
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Cancer Biology

End-synapsis Assay Authors:  Jessica Cottarel and Patrick Calsou, date: 09/20/2013, view: 3752, Q&A: 0
Many environmental agents induce double-strand breaks (DSBs) in DNA. Unrepaired or improperly repaired DSBs can lead to cell death or cancer. Nonhomologous end joining is the primary DNA double-strand break repair pathway in eukaryotes. During NHEJ pathway, several proteins recognize and bind DNA ends, bring the ends in a synaptic complex and, ...
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Isolation of Mouse Embryo Fibroblasts Authors:  Marian E Durkin, Xiaolan Qian, Nicholas C Popescu and Douglas R Lowy, date: 09/20/2013, view: 7271, Q&A: 0
Preparation of primary cultures of embryo fibroblasts from genetically engineered mouse strains can provide a valuable resource for analyzing the consequences of genetic alterations at the cellular level. Mouse embryo fibroblasts (MEFs) have been particularly useful in cancer research, as they have facilitated the identification of the genetic ...
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In vivo Chick Chorioallantoic Membrane (CAM) Angiogenesis Assays Authors:  Zhenguo Chen, Zhihua Wen and Xiaochun Bai, date: 09/20/2013, view: 16573, Q&A: 3
Angiogenesis is the process of formation of new blood vessels from pre-existing vessels or endothelial cell progenitors. It plays a crucial role in tumor growth and metastasis. Tumor angiogenesis have been widely studied as an important target for suppressing tumor growth and metastasis. Here, we describe an in vivo chick embryo ...
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Homologous Recombination Assay Authors:  Yvan Canitrot and Didier Trouche, date: 09/20/2013, view: 6950, Q&A: 0
Repair of double strand break by homologous recombination was examined using U2OS cells or RG37 cells harbouring specific substrate developed by Puget et al. (2005) and Dumay et al. (2006), respectively, to measure the repair of DNA double strand breaks by homologous recombination. The substrate is composed of two inactive copies ...
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Neutral Comet Assay Authors:  Elisa Boutet-Robinet, Didier Trouche and Yvan Canitrot, date: 09/20/2013, view: 10383, Q&A: 1
The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding ...
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Cell Biology

ROR1 Flow Cytometry Authors:  Vincent T. Bicocca and Jeffrey W. Tyner, date: 09/20/2013, view: 3308, Q&A: 0
ROR1 is a receptor tyrosine kinase family member studied for its roles in development and cancer. Here we describe a protocol for analysis of ROR1 surface expression in acute lymphoblastic leukemia immortalized cell lines by flow cytometry.
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Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation Authors:  Josse van Galen and Julia von Blume, date: 09/20/2013, view: 6191, Q&A: 0
This is a protocol to extract intact Golgi Membranes from HeLa cells using sucrose gradient centrifugation. This extraction is very useful for several applications including pull-down of Golgi membrane proteins, electron microscopy and reconstitution of protein transport into an isolated system. Protocol adapted from Balch et al. (1984).
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Immunology

Flow Cytometric Analysis of Calcium Influx Assay in T cells Author:  Sun-Hwa Lee, date: 09/20/2013, view: 7955, Q&A: 0
Calcium influx is one of the key signaling events upon stimulation of T cell receptors (TCR) and plays an important role for T cell activation, proliferation, and differentiation. Phorbol myristate acetate (PMA) and calcium ionophore ionomycin are commonly utilized as stimulants in a variety of immunologic assays including T cell activation. PMA ...
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Microbiology

Choline Uptake Assay in Bacterial Cells Authors:  Lucas Bukata, Claudia K. Herrmann and Diego J. Comerci, date: 09/20/2013, view: 4088, Q&A: 0
Choline is a methylated nitrogen compound that is widespread in nature. It is a precursor of several metabolites that perform numerous biological functions and it is predominantly used for the synthesis of essential lipid components of the cell membranes. Since there is no evidence that prokariotes can synthesize choline de novo and ...
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Isolating RNA from the Soil Authors:  Jacqueline M Chaparro and Jorge M Vivanco, date: 09/20/2013, view: 4716, Q&A: 0
Next generation sequencing has allowed for the analysis and ability to identify the microbial communities present in the environment. While DNA extraction from environments (such as soil) have provided a wealth of knowledge regarding microbial communities there are drawbacks that one encounters when using DNA as opposed to RNA. RNA allows for the ...
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Measurement of Extracellular Ca2+ Influx and Intracellular H+ Efflux in Response to Glycerol and PEG6000 Treatments Authors:  Tao Li and Baodong Chen, date: 09/20/2013, view: 4320, Q&A: 0
The characteristics of Ca2+ and H+ fluxes may reflect the activities of aquaporins, as the up-regulation of aquaporin activities is directly associated with the decrease in cytoplasmic H+ concentration and increase in cytoplasmic Ca2+ concentration. The higher aquaporin activities can protect cells ...
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Molecular Biology

Chromatin Immunoprecipitation (ChIP), Streptavidin and ATP-agarose Mediated Pull-down Analyses Authors:  Cheng-Der Liu, Ya-Lin Chen, Yi-Ly Min, Bo Zhao, Chi-Ping Cheng, Myung-Soo Kang, Shu-Jun Chiu, Elliott Kieff and Chih-Wen Peng, date: 09/20/2013, view: 6302, Q&A: 0
Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) induces expression of both viral and cellular genes in virus infected B cells by mimicking activated Notch receptors (Notch-IC) that mediate transcription activation through binding to the repressing domain of the recombining binding protein suppressor of hairless (RBP-Jκ). In general, chromatin ...
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Neuroscience

Rat Model of Chronic Midthoracic Lateral Hemisection Author:  Victor Arvanian, date: 09/20/2013, view: 5366, Q&A: 0
Although most spinal cord injuries (SCI) are anatomically incomplete, only limited functional recovery has been observed in people and rats with partial lesions. To address why surviving fibers cannot mediate more complete recovery, it is important to evaluate the physiological and anatomical status of spared fibers. These experiments require use ...
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Plant Science

Analysis of Flower Cuticular Waxes and Cutin Monomers Authors:  Anna Smirnova, Jana Leide and Markus Riederer, date: 09/20/2013, view: 4141, Q&A: 0
Here we describe procedures for the flower cuticular waxes extraction, modification and subsequent qualitative and quantitative analysis by gas-chromotography-mass spectrometry (GC-MS) and gas-chromotography with flame ionization detector (GC-FID), accordingly. To characterize flower cutin monomers two experimental setup are described: (i) ...
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Fluorescence Measurement of Postharvest Physiological Deterioration (PPD) in Cassava Storage Roots Authors:  Jia Xu, John K. Fellman and Peng Zhang, date: 09/20/2013, view: 6043, Q&A: 1
Cassava (Manihot esculenta Crantz) is a perennial root crop in the tropics. Within 24-72 hours of harvest the storage roots deteriorate rapidly, thereby necessitating their prompt processing or consumption. Postharvest physiological deterioration (PPD) of cassava storage roots is the result of a rapid oxidative burst, which leads to ...
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Stem Cell

iPS Cell Induction from Human Non-T, B cells from Peripheral Blood Author:  Keisuke Okita, date: 09/20/2013, view: 4210, Q&A: 0
The generation of iPS cells gives an opportunity to use patient-specific somatic cells which are a valuable source for disease modeling and drug discovery. To promote these studies, it is important to make iPS cells from easily accessible and less invasive tissues like blood. Here, we describe the basic method to generate human iPS cells from ...
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