By Kermit L. Carraway (Editor), Carolie A. Carothers Carraway (Editor)
This ebook offers descriptions of experimental equipment in study at the cytoskeleton and its relationships to signaling and phone law. hence, it bridges energetic and fertile components of study. the focal point is directed really in the direction of tools which benefit from contemporary advances in molecular biology, microscopy and immunological assays. A moment emphasis is on equipment for realizing dynamic adjustments in cells. a 3rd emphasis is at the formation and turnover of macromolecular and supramolecular complexes, that are so very important in riding cellphone law and the habit of cytoskeletal components. a mixture of sensible recommendation and unique protocols should still make this publication precious for either beginner and skilled staff in those burgeoning fields.
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Additional resources for Cytoskeleton: Signalling and Cell Regulation: A Practical Approach
Clones are grown, and part is used to isolate the phagemid to sequence the relevant portion of the mutant gene. The remainder is used for rescue to produce recombinant phages that are also assayed for their capability to interact with the ligand in a modified ELISA. , submitted). Although one can coat actin directly to the ELISA plates in good yield, only a small fraction remains functional. We have good experience with coating 24 2: Assaying binding and covalent modifications of cytoskeletal proteins Figure 7.
1). Actin and many components of the yeast actin cytoskeleton are highly homologous to cytoskeletal proteins of higher eukaryotes. Another advantage of yeast is the relative simplicity of the actin cytoskeleton. For example, actin and most of the actin-binding proteins are represented by a single isoform. The genetics of yeast is well developed. The genome of yeast has been sequenced, which simplifies the search for new actin-binding proteins, either by homology searches or by testing the phenotypes of mutants with targeted ORF disruptions.
4. Equipment and reagents • . « • Microcentrifuge Reagents for Bradford assay Equipment and reagents for Western blots Protease inhibitors . 0 • 5 mg/ml saponin Method 1. Grow a 25 ml yeast culture to ~ 5 x 106 cells/ml. 2. Collect the cells by centrifugation. 0 ml of cold 1 x MKEI with protease inhibitors. 38 3: Methods to study actin assembly and dynamics in yeast 3. Pellet the cells in a microcentrifuge for 2 sec, aspirate the supernatant, and freeze immediately in liquid nitrogen. 4. Thaw the frozen pellet at RT, add an equal volume of 1 x MKEI (about 100 ul) with protease inhibitors, and freeze again.