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 JoVE Biology

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

1Department of Physiology, Jules Stein Eye Institute and Howard Hughes Medical Institute, University of California, Los Angeles


JoVE 50169

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

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 JoVE Biology

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington


JoVE 3059

An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

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 JoVE Neuroscience

Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

1Pharmacology & Chemical Biology Department, University of Pittsburgh School of Medicine


JoVE 51365

Here we demonstrate the use of fluorescent Alexa dye coupled to α-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds α-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

 JoVE Biology

A Protocol for Computer-Based Protein Structure and Function Prediction

1Center for Computational Medicine and Bioinformatics, University of Michigan, 2Center for Bioinformatics and Department of Molecular Bioscience, University of Kansas


JoVE 3259

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.

 JoVE Biology

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

1Department of Chemical and Biological Engineering, Princeton University


JoVE 50476

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (http://www.proteinwisdom.org). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

 JoVE Chemistry

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

1Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University


JoVE 51383

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its ε subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with ε’s inhibitory C-terminal domain.

 JoVE Biology

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


JoVE 51206

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.

 JoVE Chemistry

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

1School of Life Sciences, University of Nottingham


JoVE 52142

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.

 JoVE Biology

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

1Department of Biochemistry, Purdue University


JoVE 51418

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.

 JoVE Biology

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

1Physical Biosciences Division, Lawrence Berkeley National Laboratory


JoVE 51715

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

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