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Biochemistry

Determination of Pseudokinase-ligand Interaction by a Fluorescence-based Thermal Shift Assay Authors:  Isabelle S Lucet, Joanne M. Hildebrand, Peter E. Czabotar, Jian-Guo Zhang, Nicos A. Nicola, John Silke, Jeffrey J. Babon and James M. Murphy, date: 06/05/2014, view: 6920, Q&A: 0
This protocol describes a robust technique for the measurement of pseudokinase-ligand interaction by a fluorescence-based Thermal Shift Assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulators of signal transduction and may therefore represent a novel class of drug targets. Unlike kinases, the catalytic ...
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Small-scale Triton X-114 Extraction of Hydrophobic Proteins Authors:  Yuzuru Taguchi and Hermann M. Schätzl, date: 06/05/2014, view: 9847, Q&A: 0
Here we introduce a protocol for Triton X-114 extraction which we used in our recently-published paper (Taguchi et al., 2013). It is a versatile method to concentrate or partially purify hydrophobic proteins. The presented protocol is based on the protocol published by Bordier (Bordier, 1981) but more simplified and down-scaled for more ...
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Protocol for Preparation of Nuclear Protein from Mouse Lungs Authors:  Zuoxiang Xiao and Qun Jiang, date: 06/05/2014, view: 6970, Q&A: 0
This protocol describes how to extract nuclear protein from mouse lungs, including tissue preparation, stepwise lysis of cells and centrifugal isolation of nuclear protein fraction. This is an efficient method to get comparable nuclear protein extracts from lung tissues.
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Cell Biology

Small-scale Subcellular Fractionation with Sucrose Step Gradient Authors:  Yuzuru Taguchi and Hermann M. Schätzl, date: 06/05/2014, view: 8600, Q&A: 0
Here, we introduce the protocol for small-scale and simple subcellular fractionation used in our recent publication (Taguchi et al., 2013), which uses homogenization by passing through needles and sucrose step-gradient.

Subcellular fractionation is a very useful technique but usually a large number of cells are required. ...
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Novel Method for Site-specific Induction of Oxidative DNA Damage to Study Recruitment of Repair Proteins to Heterochromatin and Euchromatin Authors:  Leizhen Wei, Satoshi Nakajima, Arthur S. Levine and Li Lan, date: 06/05/2014, view: 4939, Q&A: 0
ROS-induced DNA damage is repaired in living cells within a temporal and spatial context, and chromatin structure is critical to a consideration of DNA repair processes in situ. It’s well known that chromatin remodeling factors participate in many DNA damage repair pathways, indicating the importance of chromatin remodeling in ...
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Immunology

Identification of Helminth-induced Type 2 CD4+ T Cells and ILC2s Authors:  Mario M. Zaiss and Kendle M. Maslowski, date: 06/05/2014, view: 4484, Q&A: 0
After activation, T cells differentiate into different T helper (Th) subsets, namely Th1, Th2, and Th17. These different Th subsets are associated with the production of particular cytokines endowing them with different functions. In immunity against helminth infections the Th2 cell subset plays an important role. Th2 cells typically produce IL-4, ...
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In vitro Inflammasome Assay Authors:  Mario M. Zaiss and Kendle M. Maslowski, date: 06/05/2014, view: 6891, Q&A: 0
Innate immune cells sense pathogen and danger-associated molecular patterns (PAMPs and DAMPs) through a range of innate immune pattern recognition receptors (PRRs). One type of PRRs are the Nod-like receptors (NLRs), which form inflammasomes; a molecular platform required for the recruitment and activation of Caspase-1, which in turn cleaves and ...
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Protocol for Macrophage Depletion from Mice Authors:  Zuoxiang Xiao and Qun Jiang, date: 06/05/2014, view: 9837, Q&A: 0
Macrophage depletion has been used extensively to study autoimmune disease and more recently in tumor models. The clodronate-containing liposomes will be recognized as foreign particles and get engulfed by macrophages upon dosing into the animal by the chosen routes. Consequently, macrophages that have engulfed liposomes will all be destroyed by ...
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Microbiology

Preparation of Parasite Protein Extracts and Western Blot Analysis Authors:  Arlett Heiber and Tobias Spielmann, date: 06/05/2014, view: 13277, Q&A: 0
In order to prepare protein extracts of Plasmodium falciparum blood stages for western blot analysis, infected red blood cells (iRBC) need to be separated from uninfected red blood cells (uRBC) which make up the bulk of the parasite culture. Depending on the localisation of the parasite protein of interest, different methods are available ...
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Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites Authors:  Arlett Heiber, Silke Retzlaff and Tobias Spielmann, date: 06/05/2014, view: 4972, Q&A: 0
This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission ...
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Purification and Structural Analysis of QS-inhibiting Compounds from Staphylococcus delphini Authors:  Ya-Yun Chu, Mulugeta Nega and Friedrich Götz, date: 06/05/2014, view: 4213, Q&A: 0
The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we present a purification protocol for molecules excreted by a group of Gram-positive zoonotic pathogen bacteria, the ‘Staphylococcus intermedius group’, that suppress the ...
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Infant Rabbit Colonization Competition Assays Authors:  Yang Fu and John J. Mekalanos, date: 06/05/2014, view: 5259, Q&A: 0
Enteric pathogenic bacteria such as Vibrio cholerae and enteropathogenic Escherichia coli (E. coli) cause life-threatening diarrheal diseases that have afflicted humans for centuries. Understanding the effectors required for intestinal colonization is very important to research on bacteria pathogenesis, and is also ...
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Intracellular Glycogen Assays Authors:  Yong Jun Goh and Todd R. Klaenhammer, date: 06/05/2014, view: 7005, Q&A: 0
Glycogen, a soluble multi-branched glucose homopolysaccharide, is composed of chains of α-1,4-linked glucose residues interconnected by α-1,6-linked branches. The classical biosynthetic pathway involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyltransferase (GlgC or GlgCD), glycogen synthase (GlgA) and branching enzyme (GlgB). ...
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Stem Cell

Competitive Bone-marrow Transplantations Authors:  Maria Maryanovich and Atan Gross, date: 06/05/2014, view: 11231, Q&A: 0
Competitive bone marrow transplantation assay measures multi-lineage reconstitution of hematopoiesis in irradiated transplant recipient mice. Thus this assay is routinely used to determine haematopoietic stem and progenitor cells (HSPCs) functionality in vivo. The principle of the method is to transplant bone marrow donor cells (derived ...
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