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Protocol
Locomotor Assays in Drosophila Larvae and Adult Flies
Brain defects often lead to motor dysfunctions in humans. Drosophila melanogaster has been one of the most useful organisms in the study of neuronal biology due to its similarities with humans and has contributed...
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Protocol
High-Throughput Base Editing-Mediated Artificial Evolution Streamlines Trait Gene Identification in Rice
Base Editing-Mediated Gene Evolution (BEMGE) method develops novel rice germplasms with mutations in any endogenous gene through employing both Cas9n-based cytosine and adenine base editors combined with an sg...
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Protocol
Adoptive Transfer of Cryptococcus neoformans-Specific CD4 T-Cells to Study Anti-fungal Lymphocyte Responses In Vivo
CD4 T-cells are important for long-term control and clearance of several fungal infections in humans, particularly those caused by Cryptococcus species. Understanding the mechanisms underlying protective T-cell i...
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Protocol
Primary Intestinal Fibroblasts: Isolation, Cultivation, and Maintenance
Intestinal fibroblasts maintain homeostasis and contribute to inflammatory responses and the development of cancer. Intestinal fibroblasts express pattern recognition receptors which can mount an immune respon...
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Protocol
Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry
Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an in...
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Protocol
Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins
Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no...
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Protocol
Monitoring Mitochondrial Perturbations During Infection
Mitochondria are pivotal organelles in the cell that regulate a myriad of cellular functions, which eventually govern cellular physiology and homeostasis. Intriguingly, microbial infection is known to trigger ...
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Protocol
Implementation of 3D Multi-Color Fluorescence Microscopy in a Quadruple Trap Optical Tweezers System
Recent advances in the design and measurement capabilities of optical tweezers instruments, and especially the combination with multi-color fluorescence detection, have accommodated a dramatic increase in the ...
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Protocol
One-Dimensional STED Microscopy in Optical Tweezers
Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that the...
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Protocol
Rational Design and Construction of Active-Site Labeled Enzymes
With a growing amount of structural information of proteins, deciphering the linkage between the structure and function of these proteins is the next important task in structural genomics. To characterize the ...
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Protocol
An Approach to Monitor Exocytosis in White Adipocytes
Exocytosis, the fusion of vesicles with the plasma membrane, can be measured with the patch-clamp technique as increases in membrane capacitance. Here we provide detailed information on how to monitor white ad...
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Protocol
Expression Screening of Human Integral Membrane Proteins Using BacMam
This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected an...
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Protocol
Studying Protein–DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry
Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural res...
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Protocol
Biolistic DNA Delivery in Turfgrass Embryonic Callus Initiated from Mature Seeds
We describe a protocol for the establishment and preparation of cree** bentgrass (Agrostis stolonifera L.) cultivar “Penn A-4” embryonic calli, biolistic transformation, selection, and regeneration of transgeni...
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Protocol
The Multiparametric Analysis of Mitochondrial Dynamics in T Cells from Cryopreserved Peripheral Blood Mononuclear Cells (PBMCs)
The analysis of mitochondrial dynamics within immune cells allows us to understand how fundamental metabolism influences immune cell functions, and how dysregulated immunometabolic processes impact biology and...
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Protocol
Functional Genomics in the Study of Metabolic Pathways in Medicago truncatula: An Overview
In addition to its value as a model system for studies on symbiotic nitrogen fixation, Medicago truncatula has recently become an organism of choice for dissection of complex pathways of secondary metabolism. Thi...
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Protocol
Screening for Secretion of the Type VI Secretion System Protein Hcp by Enzyme-Linked Immunosorbent Assay and Colony Blot
The bacterial type VI secretion system (T6SS) is a secretory apparatus encoded by many Gram-negative bacteria. The T6SS facilitates the secretion and injection of toxic effector proteins into host cells, provi...
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Protocol
Conformational Dynamics and Interactions of Membrane Proteins by Hydrogen/Deuterium Mass Spectrometry
Hydrogen/deuterium exchange associated with mass spectrometry has been recently used to characterize the dynamics and the interactions of membrane proteins. Here we describe experimental workflow enabling loca...
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Protocol
Cloning and Characterization of Sirtuin3 (SIRT3)
Mitochondria play a pivotal role in maintaining cellular homeostasis and regulating longevity. SIRT3 is a mitochondrial sirtuin mediating the deacetylation of various metabolic and antioxidant enzymes, in turn...
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Protocol
Optogenetic Control of Fibroblast Growth Factor Receptor Signaling
FGFR1 is a member of the fibroblast growth factor family, which controls diverse cellular functions such as cell proliferation, migration, and differentiation. OptoFGFR1, an optogenetic method to modulate the ...
