Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.
Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
1Language and Genetics Department, Max Planck Institute for Psycholinguistics, 2Donders Institute for Brain, Cognition and Behaviour
Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.
Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters
1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Department of Molecular Biology, Princeton University, 3Division of Chemistry and Chemical Engineering, California Institute of Technology
This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.
Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford
Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.
1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven and Leuven Institute for Neuroscience and Disease (LIND)
Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.
Published September 18, 2013. Keywords: Cellular Biology, biology (general), biochemistry, bioengineering (general), LRRK1, LRRK2, metabolic labeling, 32P orthophosphate, immunoprecipitation, autoradiography
Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins
1School of Life Sciences, University of Nottingham
Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
1Lawrence Berkeley National Laboratory, The Molecular Foundry
More than half of proteins are small proteins (molecular mass <200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.
1Department of Biochemistry, Purdue University
Drosophila tissues often contain a heterogeneous mixture of cell types. To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.
DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems
1Physical Biosciences Division, Lawrence Berkeley National Laboratory
This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.
Published July 21, 2014. Keywords: Genetics, DNA-Affinity-Purified-chip, response regulator, transcription factor binding site, two component system, signal transduction, Desulfovibrio, lactate utilization regulator, ChIP-chip
1Department of Physics and Astronomy, VU University Amsterdam
This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.