Clark Matrix Metalloproteinase Protocols

Erscheinungsjahr 2008
ISBN: 978-1-59259-046-9
Verlag: Humana Press
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band 151, 545 Seiten, eBook

Reihe: Methods in Molecular Biology

Research in the matrix metalloproteinase field began with the demonstration by Gross and Lapière, in 1962, that resorbing tadpole tail expressed an enzyme that could degrade collagen gels. These humble beginnings have led us to the elucidation of around twenty distinct vertebrate MMPs, along with a variety of homologs from such diverse organisms as sea urchin, plants, nematode worm, and bacteria. This, coupled with four known specific inhibitors of MMPs, the TIMPs, gives a complex picture. Part I of Matrix Metalloproteinase Protocols provides the reader with a selective overview of the MMP arena, and a chance to come to grips with where the field has been, where it is, and where it is going. I hope that this complements all of the methodology that comes later. Part II presents the reader with a diverse set of methods for the expression and purification of MMPs and TIMPs, bringing together the long and often hard-earned experience of a number of researchers. Part III allows the reader to detect MMPs and TIMPs at both the protein and mRNA level, whereas Part IV gives the ability to assay MMP and TIMP activities in a wide variety of circumstances.

Zielgruppe

Research

Autoren/Hrsg.

Clark, Ian M.

Inhaltsverzeichnis

MMPs and TIMPs—An Overview of the Field.- MMPs and TIMPs.- Strategies for Cloning New MMPs and TIMPs.- Structural Studies on MMPs andTIMPs Wolfram Bode and Klaus Maskos.- Matrix MetaIIoproteinase Substrate Binding Domains, Modules and Exosites.- The Matrix Metalloproteinase (MMP) and Tissue Inhibitor of Metalloproteinase (TIMP) Genes.- Models for Gain-of-Function and Loss-of-Function of MMPs.- Expression and Purification of MMPs and TIMPs.- Expression of MMPs andTIMPs in Mammalian Cells.- Expression of Recombinant Matrix Metalloproteinases in Escherichia coli.- Expression of Human Collagenase I (MMP-1) andTIMP-1 in a Baculovirus-Based Expression System.- Expression of Recombinant Matrix Metalloproteinases in Yeast.- Expression of Recombinant Membrane-Type MMPs.- Refolding of TIMP-2 from Escherichia coli Inclusion Bodies.- Expression and Refolding of Full-Length HumanTIMP-1.- Purification of MMPs and TIMPs.- Detection of MMPs and TIMPs.- Monitoring MMP andTIMP mRNA Expression by RT-PCR.- Measuring Transcription of Metalloproteinase Genes.- In Situ Hybridization for Metalloproteinases and Their Inhibitors.- Use of EIA to Measure MMPs and TIMPs.- Immunohistochemistry of MMPs and TIMPs.- Detecting Polymorphisms in MMP Genes.- 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, andTIMPs.- In Situ Zymography.- Detection of Focal Proteolysis Using Texas-Red-Gelatin.- Antibodies to MMP-Cleaved Aggrecan.- Cartilage Proteoglycan Release Assay.- Immunoassay for Collagenase-Mediated Cleavage of Types I and II Collagens.- Collagen Degradation Assays.- Invasion Assays and Matrix Metalloproteinases.- Using FluorogenicPeptide Substrates to Assay Matrix Metalloproteinases.- Kinetic Analysis of the Inhibition of Matrix Metalloproteinases by Tissue Inhibitor of Metalloproteinases (TIMP).- Assaying Growth Factor Activity of Tissue Inhibitors of Metalloproteinases.