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Biochemistry

Development and Application of a Fully Blind Flexible Peptide-protein Docking Protocol, pepATTRACT Authors:  Christina EM Schindler, Sjoerd J. de Vries and Martin Zacharias, date: 06/05/2016, view: 2094, Q&A: 0
Peptide-mediated interactions are involved in many signaling and regulatory pathways as well as the DNA replication machinery and are linked to many pathological disorders. Many research groups are currently working towards a more detailed understanding of these important interactions by characterizing the 3D complex structures with experimental ...
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Cancer Biology

In vitro mTORC1 Kinase Assay for Mammalian Cells Protocol Authors:  Kayleigh Margaret Dodd and Andrew Robert Tee, date: 06/05/2016, view: 2859, Q&A: 0
Historically, mechanistic target of rapamycin (mTOR) was purified from mammalian cells using mild nonionic detergents such as NP-40 and or Triton-X100 that resulted in dissociation of core regulatory components essential for its native kinase activity. Consequently, these older kinase assays required MnCl2 to artificially enhance the ...
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In vitro Tumor Cell Migration Assay Using ThinCertsTM (Transwells) Author:  Marc A. Schneider, date: 06/05/2016, view: 4092, Q&A: 0
The high migration rate of tumor cells often results in poor prognosis for the survival of the patients. Here, we describe a protocol to measure the migration of cells using a quantitative assay. The relative tumor cell migration was measured using ThinCertsTM cell culture inserts and a lactate dehydrogenase (LDH) assay to quantify the ...
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Cell Biology

Isolating Taste Buds and Taste Cells from Vallate Papillae of C57BL/6J Mice for Detecting Transmitter Secretion Authors:  Anthony Y. Huang and Sandy Y. Wu, date: 06/05/2016, view: 2013, Q&A: 0
Mouse is a well-accepted model for studying taste bud function. Mice readily detect and respond to taste substances that humans consider to have sweet, bitter, salty, sour and umami taste qualities. A great deal of recent research on taste receptors is based on this species. Live mice are needed for these experiments because no alternative in ...
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Aorta Atherosclerosis Lesion Analysis in Hyperlipidemic Mice Authors:  Sarajo Mohanta, Changjun Yin, Christian Weber, Desheng Hu and Andreas J. R. Habenicht, date: 06/05/2016, view: 4164, Q&A: 0
Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries. Apolipoprotein E-deficient (ApoE-/-) mice are used as experimental models to study human atherosclerosis. ApoE-/- mice are constitutively hyperlipidemic and develop intima plaques that resemble human plaques. Various issues including ...
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Immunology

Hemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies Authors:  Ying Wu, MyungSam Cho, David Shore, Manki Song, JungAh Choi, Tao Jiang, Yong-Qiang Deng, Melissa Bourgeois, Lynn Almli, Hua Yang, Li-Mei Chen, Yi Shi, Jianxu Qi, An Li, Kye Sook Yi, MinSeok Chang, Jin Soo Bae, HyunJoo Lee, JiYoung Shin, James Stevens, SeoungSuh Hong, Cheng-Feng Qin, George F. Gao, Shin Jae Chang and Ruben O. Donis, date: 06/05/2016, view: 5000, Q&A: 0
Heamagglutination is inhibited when antibodies are present because antibodies to the influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. Human monoclonal antibodies generated from single human B cells were tested to characterize their ...
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Micro Neutralization (MN) Assay of Influenza Viruses with Monoclonal Antibodies Authors:  Ying Wu, MyungSam Cho, David Shore, Manki Song, JungAh Choi, Tao Jiang, Yong-Qiang Deng, Melissa Bourgeois, Lynn Almli, Hua Yang, Li-Mei Chen, Yi Shi, Jianxu Qi, An Li, Kye Sook Yi, MinSeok Chang, Jin Soo Bae, HyunJoo Lee, JiYoung Shin, James Stevens, SeoungSuh Hong, Cheng-Feng Qin, George F. Gao, Shin Jae Chang and Ruben O. Donis, date: 06/05/2016, view: 2660, Q&A: 0
The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to neutralize virus infectivity. The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses. This protocol is to measure the ability of human monoclonal ...
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Preparation of Single Cell Suspensions from Mouse Aorta Authors:  Desheng Hu, Changjun Yin, Sarajo Mohanta, Christian Weber and Andreas J. R. Habenicht, date: 06/05/2016, view: 3443, Q&A: 0
Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia ...
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Microbiology

MNase Digestion for Nucleosome Mapping in Neurospora Authors:  Cigdem Sancar, Gencer Sancar and Michael Brunner, date: 06/05/2016, view: 2333, Q&A: 0
Digestion of chromatin by micrococcal nuclease MNase followed by high throughput sequencing allows us to determine the location and occupancy of nucleosomes on the genome. Here in this protocol we have described optimized conditions of MNase digestion of filamentous fungus Neurospora crassa chromatin without a requirement of a nuclear ...
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Neuroscience

Sensitive Assessment of Hippocampal Learning Using Temporally Dissociated Passive Avoidance Task Authors:  Andrew L. Eagle, Hongbing Wang and Alfred J. Robison, date: 06/05/2016, view: 2173, Q&A: 0
The temporally dissociated passive avoidance (TDPA) paradigm is a variant of passive avoidance testing, and allows for more sensitive investigation of mild impairments in avoidance learning. Passive avoidance learning measures the latency to enter a “dark” context in which an aversive stimulus (foot shock) has been previously experienced using a ...
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Sucrose Preference Test to Measure Stress-induced Anhedonia Authors:  Andrew L. Eagle, Michelle Mazei-Robison and Alfred J. Robison, date: 06/05/2016, view: 7171, Q&A: 0
The 2-bottle choice procedure for assessing sucrose preference is a useful test to investigate anhedonia (i.e., inability to feel pleasure) in laboratory rodents, particularly in stress-based models of depression. The 2-bottle choice procedure allows for a comparison between behavioral preference for sucrose solution in drinking water ...
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In utero Electroporation of Mouse Cerebellar Purkinje Cells Author:  Yukari H. Takeo, date: 06/05/2016, view: 3542, Q&A: 0
In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and ...
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Plant Science

Bioassays to Investigate the Effects of Insect Oviposition on a Plant’s Resistance to Herbivores Authors:  Michele Bandoly and Anke Steppuhn, date: 06/05/2016, view: 3316, Q&A: 0
Plants respond to herbivory with diverse defence responses (Schoonhoven et al., 2005). Many herbivorous insects deposit their eggs on their host plants before their larvae start to feed. Thus, plants could use insect eggs as a signal to increase their resistance to herbivores. Here, we report experimental procedures to explore whether and ...
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Differential and Simultaneous Visualization of Cells and Airspaces in Plant Leaves Authors:  Takashi Kawase, Shigeo S. Sugano, Tomoo Shimada and Ikuko Hara-Nishimura, date: 06/05/2016, view: 2050, Q&A: 0
This protocol is to differentially and simultaneously visualize both cells and airspaces in intact leaves and to create 3D structures of cells and airspaces from confocal images using the software DSLT_Demo (https://github.com/nslab2000/DSLT). Leaves stained with Nile Red in ...
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Quantification of the Adhesion Strength between Peroxisomes and Chloroplasts by Femtosecond Laser Technology Authors:  Yoichiroh Hosokawa, Takanori Iino, Kazusato Oikawa, Shoji Mano, Kenji Yamada and Mikio Nishimura, date: 06/05/2016, view: 2528, Q&A: 0
This is the detailed protocol to quantify adhesion strength between peroxisomes and chloroplasts in plant leaf palisade mesophyll cells described by Oikawa et al. (2015). The quantification was performed by utilizing local explosion induced by focusing femtosecond laser pulses into a mesophyll cell under a confocal microscope. When an ...
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