Matrix Metalloproteinase Protocols

by Clark, Ian M.; Young, David A.; Rowan, Andrew D.

• ISBN13: 9781603272988
• ISBN10: 1603272984
• Edition: 2nd
• Format: Hardcover
• Copyright: 2010-03-24
• Publisher: Humana Pr Inc

Summary

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.

Table of Contents

Preface	p. v
Contributors	p. ix
MMPs and TIMPs: an Overview	p. 1
Metalloproteases and the Degradome	p. 3
Mouse Models of MMP and TIMP Function	p. 31
Expression and Purification of MMPs and TIMPs	p. 53
Expression of Recombinant MMP-28 in Mammalian Cells	p. 55
Expression of Recombinant Matrix Metalloproteinases in Escherichia coli	p. 67
Expression of Recombinant ADAMTS in Insect Cells	p. 83
Expression and Purification of Membrane-Type MMPs	p. 99
Refolding of TIMP-2 from Escherichia coli Inclusion Bodies	p. 111
Purification of MMPs and TIMPs	p. 123
Detection of MMPs and TIMPs	p. 157
Real-Time PCR Expression Profiling of MMPs and TIMPs	p. 159
Analysis of the Degradome with the CLIP-CHIP™ Microarray	p. 175
In Situ Hybridization for Metalloproteinases and Their Inhibitors	p. 195
Immunohistochemistry of MMPs and TIMPs	p. 211
Single Nucleotide Polymorphism Genotyping in MMP Genes: The 5' Nuclease Assay	p. 221
Assay of MMP and TIMP Activities	p. 231
Methods for Studying Activation of Matrix Metalloproteinases	p. 233
Assay of Matrix Metalloproteinases Against Matrix Substrates	p. 245
Zymography and Reverse Zymography for Detecting MMPs and TIMPs	p. 257
In Situ Zymography	p. 271
Near-Infrared Optical Proteolytic Beacons for In Vivo Imaging of Matrix Metalloproteinase Activity	p. 279
Neoepitope Antibodies Against MMP-Cleaved and Aggrecanase-Cleaved Aggrecan	p. 305
In Vitro Model of Cartilage Degradation	p. 341
Immunoassays for Collagenase-Mediated Cleavage of Type I and II Collagens	p. 349
Collagen Degradation Assays	p. 367
Analysis of MMP-Dependent Cell Migration and Invasion	p. 379
Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases	p. 393
Kinetic Analysis of the Inhibition of Matrix Metalloproteinases: Lessons from the Study of Tissue Inhibitors of Metalloproteinases	p. 435
Identification of Cellular MMP Substrates Using Quantitative Proteomics: Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ)	p. 451
Mechanism-Based Profiling of MMPs	p. 471
Subject Index	p. 489
Table of Contents provided by Ingram. All Rights Reserved.