{"@context":"http://iiif.io/api/presentation/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/manifest.json","@type":"sc:Manifest","label":"Developing Tools for Cavin-1 Purification and Binding Studies with Caveolin-1","metadata":[{"label":"dc.description.sponsorship","value":"This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree."},{"label":"dc.format","value":"Monograph"},{"label":"dc.format.medium","value":"Electronic Resource"},{"label":"dc.identifier.uri","value":"http://hdl.handle.net/11401/76930"},{"label":"dc.language.iso","value":"en_US"},{"label":"dc.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.abstract","value":"Caveolae are nanoscale invaginations found in the plasma membrane of mammalian cells, and are implicated in numerous essential cellular communication and transport processes. They are characterized by a coat composed of caveolin and cavin proteins. Recent studies of the caveolar coat complex suggest that cavin-1 associates with caveolin-1 during caveolae biogenesis.  However, it is not known how this binding occurs.  Studies of the spatial and physical interactions between cavin-1 and caveolin-1 can provide valuable insights into the mechanism of caveolae generation. The goal of this study was to test two different fusion tags to purify a sufficient quantity of cavin-1, for use in future binding studies with caveolin-1. We successfully generated a plasmid encoding 6His-SUMO-cavin-1 by cloning full length cavin-1 into the LIC SUMO vector 2S-T, and expressed it in BL21 (DE3) pLysS. Parameters of temperature, concentration of IPTG, detergent type, and length of incubation after IPTG-induction, were tested to optimize cavin-1 expression conditions. Immobilized metal affinity chromatography with Ni-NTA resin tested binding and release of the SUMO-tagged cavin-1. The poor protein yields from tests on 6-His-SUMO-cavin-1 underscored persistent issues of solubility, suboptimal binding to the resin, and inadequate release of purified protein. Studies on an earlier construct, GST-cavin-1, were then resumed to evaluate and maximize binding and release of GST-cavin-1 from glutathione-agarose beads. While inefficient bead binding problems remained, GST-cavin-1 yielded a more consistent recovery of the desired protein. As results suggested that enough soluble GST-cavin-1 could be obtained for binding studies, we performed a large-scale preparation of the protein for use in a diagnostic test for association with caveolin-1. We did not observe any binding in this preliminary test. The results prompted further questions about the stoichiometry of the cavin-1 and caveolin-1 complex, and highlighted the possibility of additional layers of complexity involved in this interaction."},{"label":"dcterms.available","value":"2017-09-20T16:51:27Z"},{"label":"dcterms.contributor","value":"French, Jarrod."},{"label":"dcterms.creator","value":"Siddiqui, Safa Farjand"},{"label":"dcterms.dateAccepted","value":"2017-09-20T16:51:27Z"},{"label":"dcterms.dateSubmitted","value":"2017-09-20T16:51:27Z"},{"label":"dcterms.description","value":"Department of Biochemistry and Cell Biology."},{"label":"dcterms.extent","value":"77 pg."},{"label":"dcterms.format","value":"Monograph"},{"label":"dcterms.identifier","value":"http://hdl.handle.net/11401/76930"},{"label":"dcterms.issued","value":"2015-12-01"},{"label":"dcterms.language","value":"en_US"},{"label":"dcterms.provenance","value":"Made available in DSpace on 2017-09-20T16:51:27Z (GMT). No. of bitstreams: 1\nSiddiqui_grad.sunysb_0771M_12533.pdf: 2034619 bytes, checksum: 5b594d51ced2ef7e6d220ee04b580047 (MD5)\n  Previous issue date:    1"},{"label":"dcterms.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.subject","value":"Biochemistry"},{"label":"dcterms.title","value":"Developing Tools for Cavin-1 Purification and Binding Studies with Caveolin-1"},{"label":"dcterms.type","value":"Thesis"},{"label":"dc.type","value":"Thesis"}],"description":"This manifest was generated dynamically","viewingDirection":"left-to-right","sequences":[{"@type":"sc:Sequence","canvases":[{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json","@type":"sc:Canvas","label":"Page 1","height":1650,"width":1275,"images":[{"@type":"oa:Annotation","motivation":"sc:painting","resource":{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/78%2F78%2F50%2F78785086346011009011062395472428328879/full/full/0/default.jpg","@type":"dctypes:Image","format":"image/jpeg","height":1650,"width":1275,"service":{"@context":"http://iiif.io/api/image/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/78%2F78%2F50%2F78785086346011009011062395472428328879","profile":"http://iiif.io/api/image/2/level2.json"}},"on":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json"}]}]}]}