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Full Description
Since the discovery of a collagen-degrading protease in the tadpole tail in 1962, matrix metalloproteinase research has led to the discovery of more than twenty distinct vertebrate MMPs, along with a variety of homologues from diverse organisms such as the sea urchin, plants, insects, and nematode worms. Fully updating and adding to the popular first edition, Matrix Metalloproteinase Protocols, Second Edition includes a series of state-of-the-art techniques provided by eminent experts in the field. Beginning with a brief overview of the MMP arena, from how these enzymes fit into the larger degradome to what occurs when their expression and function in the mouse is modulated, the volume continues with sections on the expression and purification of MMPs and TIMPs, the detection of MMPs and TIMPs at both the protein and mRNA level, and our ability to assay MMP and TIMP activities in a wide variety of circumstances. Written in the highly successful Methods in Molecular Biology™ series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.
Comprehensive and cutting-edge, Matrix Metalloproteinase Protocols, Second Edition is an ideal source for many of the essential laboratory techniques for both novice and seasoned researchers alike collected in one convenient volume.
Contents
MMPs and TIMPs: An Overview.- Metalloproteases and the Degradome.- Mouse Models of MMP and TIMP Function.- Expression and Purification of MMPs and TIMPs.- Expression of Recombinant MMP-28 in Mammalian Cells.- Expression of Recombinant Matrix Metalloproteinases in Escherichia coli.- Expression of Recombinant ADAMTS in Insect Cells.- Expression and Purification of Membrane-Type MMPs.- Refolding of TIMP-2 from Escherichia coli Inclusion Bodies.- Purification of MMPs and TIMPs.- Detection of MMPs and TIMPs.- Real-Time PCR Expression Profiling of MMPs and TIMPs.- Analysis of the Degradome with the CLIP-CHIP™ Microarray.- In Situ Hybridization for Metalloproteinases and Their Inhibitors.- Immunohistochemistry of MMPs and TIMPs.- Single Nucleotide Polymorphism Genotyping in MMP Genes: The 5? Nuclease Assay.- Assay of MMP and TIMP Activities.- Methods for Studying Activation of Matrix Metalloproteinases.- Assay of Matrix Metalloproteinases Against Matrix Substrates.- Zymography and Reverse Zymography for Detecting MMPs and TIMPs.- In Situ Zymography.- Near-Infrared Optical Proteolytic Beacons for In Vivo Imaging of Matrix Metalloproteinase Activity.- Neoepitope Antibodies Against MMP-Cleaved and Aggrecanase-Cleaved Aggrecan.- In Vitro Model of Cartilage Degradation.- Immunoassays for Collagenase-Mediated Cleavage of Type I and II Collagens.- Collagen Degradation Assays.- Analysis of MMP-Dependent Cell Migration and Invasion.- Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases.- Kinetic Analysis of the Inhibition of Matrix Metalloproteinases: Lessons from the Study of Tissue Inhibitors of Metalloproteinases.- Identification of Cellular MMP Substrates Using Quantitative Proteomics: Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative andAbsolute Quantification (iTRAQ).- Mechanism-Based Profiling of MMPs.
