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Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, several affinity reagents against fluorescent proteins have also been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfil their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or in living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.
Pubmed ID: 30237292
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Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
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View all literature mentionsSoftware for macromolecular model building, model completion and validation, and protein modelling using X-ray data. Coot displays maps and models and allows model manipulations such as idealization, rigid-body fitting, ligand search, Ramachandran plots, non-crystallographic symmetry and more. Source code is available.
View all literature mentionsA structure-validation web application which provides an expert-system consultation about the accuracy of a macromolecular structure model, diagnosing local problems and enabling their correction. MolProbity works best as an active validation tool (used as soon as a model is available and during each rebuild/refine loop) and when used for protein and RNA crystal structures, but it may also work well for DNA, ligands and NMR ensembles. It produces coordinates, graphics, and numerical evaluations that integrate with either manual or automated use in systems such as PHENIX, KiNG, or Coot.
View all literature mentionsCell line HeLa S3 is a Cancer cell line with a species of origin Homo sapiens (Human)
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