Protein Lipidation ProtocolsMichael H. Gelb Springer Science & Business Media, 3. 2. 2008 - Počet stran: 240 In Protein Lipidation Protocols, Michael Gelb brings together a collection of readily reproducible techniques for studying protein lipidation, the covalent attachment of lipids to proteins. These cutting-edge methods-many never published before in a "hands-on" format-deal with glycosyl phosphatidylinositol (GPI)-containing compounds, protein fatty acylation, and protein prenylation. Included are novel techniques for determining the chemical structure of GPI-anchors, for radiolabeling the prenyl groups of protein in eukaryotic cells, a tool for developing inhibitors of the protein farnesyltransferase, and for an exciting lysosomal enzyme that cleaves fatty acyl groups from proteins, the first fatty acylase discovered. Protein Lipidation Protocols offers biochemists, cell and molecular biologists, medicinal chemists, and pharmaceutical researchers state-of-the-art tools for understanding the complex biochemistry of protein lipidation, as well as catalyzing the development of many important new biopharmaceuticals, including anticancer drugs. |
Obsah
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Selection of Mammalian Cell Mutants in GPI Biosynthesis | 13 |
Analysis of the CellSurface Distribution of GPIAnchored Proteins | 23 |
Imaging Fluorescence Resonance Energy Transfer as Probe | 37 |
Proteins HRas SrcFamily Tyrosine Linases eNOS | 51 |
Analysis of Lipids in Caveolae | 61 |
Analysis of the Carbohydrate Components | 73 |
Analysis of the Lipid Moiety of GPI Anchor in the Yeast Saccharomyces | 91 |
Incorporation of Radiolabeled Prenyl Alcohols and Their Analogs into | 125 |
Subunits | 145 |
Probing the Role of HRas Lipidation for Signaling Functions in Xeno | 161 |
Fluorescence Measurement of LipidBinding Affinity and Interbilayer | 177 |
Determination of the Kinetics of Intervesicle Transfer and Transbilayer | 187 |
Metabolic Labeling of ProteinDerived Lipid Thioesters in Palmitoyl | 213 |
Fatty Acid Analysis of ProteinDerived Lipid Thioesters Isolated from | 221 |
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acceptor acetate acetonitrile acid aliquots analysis antibody assay beads Biochem Biol bovine buffer caveolae caveolin cell line centrifuge ceramide Chem chromatography Ci/mmol column concentration containing culture described in Subheading donor Dpr1 edited EDTA elution enzyme Eppendorf tubes extract farnesyl filter fluorescence fraction FRET FTase Gelb glass glycans glycosylphosphatidylinositol GPI biosynthesis GPI-anchored proteins H-Ras HPLC imaging incubated inhibitors isoprenylated kinase lipid lipidated peptide lysate lysis mammalian cells MBP-Ram2 medium metabolically labeled methanol Methods MgCl2 microcentrifuge microcentrifuge tubes microscopy Molecular mutant NaCl nitrogen NMT activity oocytes palmitoylation pellet peptide phase photobleaching plate PMSF prenylated prepared pronase Protein Lipidation Protocols purification radioactivity radiolabeled reaction reagent Reconstitution remove Resuspend room temperature sample scintillation SDS-PAGE Sigma sodium solution solvent sonicator sphingomyelin step structure subunit sucrose supernatant surface synthesis thioesters transbilayer transfer Tris-HCl Triton vesicles Vortex wash yeast µg/mL