* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DOC
Immunoprecipitation wikipedia , lookup
Circular dichroism wikipedia , lookup
Structural alignment wikipedia , lookup
Rosetta@home wikipedia , lookup
Green fluorescent protein wikipedia , lookup
List of types of proteins wikipedia , lookup
Protein domain wikipedia , lookup
Protein design wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Protein folding wikipedia , lookup
Homology modeling wikipedia , lookup
Protein structure prediction wikipedia , lookup
Protein moonlighting wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Western blot wikipedia , lookup
Protein purification wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Publication deGradFP: A System to Knockdown GFP-Tagged Proteins. JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift) ID 3644116 Author(s) Caussinus, Emmanuel; Affolter, Markus Author(s) at UniBasel Affolter, Markus; Year 2016 Title deGradFP: A System to Knockdown GFP-Tagged Proteins. Journal Methods Mol Biol Volume 1478 Pages / Article-Number 177-187 Protein depletion by genetic means, in a very general sense including the use of RNA interference [1, 2] or CRISPR/Cas9-based methods, represents a central paradigm of modern biology to study protein functions in vivo. However, acting upstream the proteic level is a limiting factor if the turnover of the target protein is slow or the existing pool of the target protein is important (for instance, in insect embryos, as a consequence of a strong maternal contribution). In order to circumvent these problems, we developed deGradFP [3, 4]. deGradFP harnesses the ubiquitin-proteasome pathway to achieve direct depletion of GFP-tagged proteins. deGradFP is in essence a universal method because it relies on an evolutionarily conserved machinery for protein catabolism in eukaryotic cells; see refs. 5, 6 for review. deGradFP is particularly convenient in Drosophila melanogaster where it is implemented by a genetically encoded effector expressed under the control of the Gal4 system. deGradFP is a ready-to-use solution to perform knockdowns at the protein level if a fly line carrying a functional GFP-tagged version of the gene of interest is available. Many such lines have already been generated by the Drosophila community through different technologies allowing to make genomic rescue constructs or direct GFP knockins: protein-trap stock collections [7, 8] ( http://cooley.medicine.yale.edu/flytrap/ , http://www.flyprot.org/ ), P[acman] system [9], MiMIC lines [10, 11], and CRISPR/Cas9-driven homologous recombination.Two essential controls of a protein knockdown experiment are easily achieved using deGradFP. First, the removal of the target protein can be assessed by monitoring the disappearance of the GFP tag by fluorescence microscopy in parallel to the documentation of the phenotype of the protein knockdown (see Note 1 ). Second, the potential nonspecific effects of deGradFP can be assessed in control fly lacking a GFP-tagged target protein. So far, no nonspecific effects of the deGradFP effector have been reported [3]. ISSN/ISBN 1940-6029 Full Text on edoc Digital Object Identifier DOI 10.1007/978-1-4939-6371-3_9 PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/27730581