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Cell Biology

TRIPLE (Insulin, Glucagon and EGFP) Immunofluorescence Staining Protocol in Pancreas Authors:  Woogyun Choi, Randal J. Kaufman and Sung Hoon Back, date: 03/05/2014, view: 8494, Q&A: 0
This protocol aims to introduce methods for immunostaining two endogenous proteins insulin and glucagon and one exogenous transgene driven EGFP in mouse pancreatic islet. The immunostaining results of insulin and glucagon indirectly tell functionality of pancreatic beta cells and alpha cells respectively. Furthermore, the protocol provides ...
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IP-Kinase Assay Author:  Pearl A. Campbell, date: 03/05/2014, view: 10825, Q&A: 0
Immunoprecipitation (IP)- Kinase assays are an invaluable tool to assess the activation status of intracellular signaling cascades within a specific cellular state and also to confirm the enzymatic activity of a specific kinase towards a putative substrate of interest. Intracellular signal transduction cascades play an important role in ...
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Microbiology

Cyclic Nucleotide (cAMP and cGMP) Assays and Capture ELISA for Quantitative Analysis of Plasmodium falciparum Blood-stage Egress Authors:  Fiona Hackett, Christine R Collins, Malcolm Strath and Michael J Blackman, date: 03/05/2014, view: 4996, Q&A: 0
Upon rupture of Plasmodium falciparum (P. falciparum) schizonts in vitro (an event known as egress), merozoites are released into the culture medium. The merozoites invade fresh red blood cells, a process that involves shedding of a microneme protein called apical membrane antigen-1 (AMA1) from the merozoite surface. ...
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Protein Sample Preparation for Proteomic Analysis in Leishmania donovani Authors:  Alexandros Alexandratos and Despina Smirlis, date: 03/05/2014, view: 5266, Q&A: 0
Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional over pre-transcriptional ...
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Co-immunoprecipitation of Flag-TLR3 or Myc-MSR1 with HCV RNA Authors:  Daisuke Yamane, Hiromichi Dansako and Stanley M. Lemon, date: 03/05/2014, view: 4761, Q&A: 0
Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize ...
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Measurement of Haemolysin Activities in Vibrio vulnificus Authors:  Hyun-Jung Lee, Jeong-A Kim, Mi-Ae Lee, Soon-Jung Park and Kyu-Ho Lee, date: 03/05/2014, view: 3857, Q&A: 0
VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. Fur repressed ...
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Generation and Screening of a Non-typeable Haemophilus influenzae Tn-seq Mutant Library Author:  Jeroen D. Langereis, date: 03/05/2014, view: 6068, Q&A: 0
The genome-wide screen Tn-seq (van Opijnen et al., 2009) is very valuable tools to identify bacterial genes with a conditionally essential function, for instance genes involved in bacterial virulence. These techniques are based on the generation of a random mutant library, which is grown in a control of challenge situation (Figure 1). The ...
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Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants Author:  Jeroen D. Langereis, date: 03/05/2014, view: 5629, Q&A: 0
Directed deletion mutants in non-typeable Haemophilus influenzae can be made by allelic exchange of the target gene with an artificial DNA construct in which an antibiotic resistance cassette is placed between two ~1,000 bp DNA sequences that are identical to the 5' and 3' flanking regions of the target gene. The artificial DNA construct ...
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Neuroscience

X-gal Staining on Adult Mouse Brain Sections Authors:  Hiroshi Kokubu and Janghoo Lim, date: 03/05/2014, view: 8993, Q&A: 0
Knowing expression patterns of given proteins is very important to understand their functions. Immunostaining analysis with specific antibodies is commonly used to identify cells or tissues expressing proteins of interest. Although this technique is regularly used, it requires high quality of specific antibodies and there is no good quality of ...
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Plant Science

Detection of Transposable Element Insertion Site Polymorphisms by Sequence-Specific Amplification Polymorphism (SSAP) Authors:  Véronique Sarilar, Paulina Martinez Palacios and Karine Alix, date: 03/05/2014, view: 5124, Q&A: 0
Transposable elements represent a major part of any eukaryotic genomes. Notably in plants they can account for more than 80% of the whole genomic sequence (such as in maize). Due to their mobility across the genome, they can act as mutagens but can also be considered as an important source of genetic diversity. It has been shown that they may be ...
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VIGS Assays Authors:  Haili Zhang and Yule Liu, date: 03/05/2014, view: 10222, Q&A: 0
Virus-induced gene silencing (VIGS) is a powerful method to study gene function in plants. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector so far. This method was originally developed by the Dinesh-Kumar's group (Liu et al., 2002) . Here, we describe a rapid and high efficient TRV-based VIGS method for ...
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Electrical Penetration Graph Recording (Whitefly) Authors:  Baiming Liu, Xuguo Zhou and Youjun Zhang, date: 03/05/2014, view: 6680, Q&A: 0
Electrical penetration graph (EPG) was first developed by Mclean and Kinsey (1964) and later modified by Tjallingii (1978 and 1985). The EPG system turns a phloem-sucking insects and its host plant into part of an electrical circuit that is completed when the insect’s mouthparts penetrate the plant. The electrical signal is amplified by an ...
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Stem Cell

Alkaline Phosphatase Staining Author:  Pearl A. Campbell, date: 03/05/2014, view: 13614, Q&A: 0
Two main features characterize pluripotent cells; self-renewal (unlimited cell division) and the ability to give rise to all cells of the adult organism. Given the recent impact of induced pluripotent stem cells (iPSCs) and ongoing use of pluripotent embryonic stem cells ESCs (ESCs) in basic discovery, drug development, and potential use for stem ...
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LT-HSC Methylcellulose Assay Author:  Marc A. Kerenyi, date: 03/05/2014, view: 9666, Q&A: 0
Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter ...
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