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Collagen : methods and protocols

Author: Irit Sagi; Afratis Nikolaos A
Publisher: New York, NY : Humana Press ; Springer, [2019] ©2019
Series: Methods in molecular biology (Clifton, N.J.), v. 1944.
Edition/Format:   eBook : Document : EnglishView all editions and formats
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Genre/Form: Electronic books
Laboratory Manual
Electronic books. 9781493990955
Laboratory manuals
Additional Physical Format: Print version:
Collagen.
New York, NY : Humana Press ; Springer, ©2019
Material Type: Document, Internet resource
Document Type: Internet Resource, Computer File
All Authors / Contributors: Irit Sagi; Afratis Nikolaos A
ISBN: 9781493990955 1493990950
OCLC Number: 1089398799
Description: 1 online resource (xi, 254 pages) : illustrations (some color)
Contents: Intro; Preface; Contents; Contributors; Part I: In Vitro Models for Collagen Characterization; Chapter 1: Cloning and Mutagenesis Strategies for Large Collagens; 1 Introduction; 2 Materials; 2.1 Polymerase Chain Reaction (PCR); 2.2 Gibson Reaction and Bacterial Transformation; 2.3 Recombinant Expression of Collagen VII; 3 Methods; 3.1 Strategy Design and Generation of DNA Fragments with Overlapping Ends; 3.2 Gibson Assembly and Bacterial Transformation; 3.3 Collagen VII Expression in HEK293 Cells; 3.4 First Assessment of Functionality of Engineered Collagen VII Variants; 4 Notes; References Chapter 2: Multi-well Fibrillation Assay as a Tool for Analyzing Crosslinking and Stabilization of Collagen1 Introduction; 2 Materials; 2.1 Collagen Isolation; 2.2 Collagen Fibrillation; 2.2.1 Fibrillation Buffer; 3 Methods; 3.1 Collagen Isolation; 3.2 Collagen Fibrillation; 4 Notes; References; Chapter 3: Production and Characterization of Chemically Cross-Linked Collagen Scaffolds; 1 Introduction; 2 Materials; 2.1 Collagen Cross-Linking and Film Preparation; 2.2 Swelling Assay; 2.3 Tensile Test; 2.4 Cell Culture; 2.5 Cell Proliferation and Morphology Assay 2.6 Metabolic Activity Assay (alamarBlue)2.7 Cell Viability Assay; 3 Methods; 3.1 Collagen Cross-Linking and Film Preparation; 3.2 Swelling Assay; 3.3 Tensile Test; 3.4 Cell Culture; 3.4.1 Scaffold Preparation; 3.4.2 DF Seeding; 3.4.3 THP-1 Seeding; 3.5 Cell Proliferation and Morphology Assay; 3.6 Cell Metabolic Activity Assay (alamarBlue); 3.7 Cell Viability Assay; 4 Notes; References; Chapter 4: A Biomimetic Model for Mineralization of Type-I Collagen Fibrils; 1 Introduction; 2 Materials; 3 Methods; 3.1 Conventional TEM Experiments; 3.1.1 Self-Assembly of Type-I Collagen Fibrils 3.1.2 Collagen Mineralization3.1.3 TEM Imaging and Mineralization Degree Calculation; 3.2 CryoTEM Experiments; 3.2.1 Self-Assembly of Type-I Collagen Extracted from Rat Tail Tendon; 3.2.2 Self-Assembly of Type-I Collagen Extracted from Horse Tendon; 3.2.3 Collagen Mineralization; 3.2.4 CryoTEM Imaging; 3.3 STORM Experiments; 3.3.1 Modification of the Laser Confocal Culture Dish (LCCD) and Self-Assembly of Type-I Collagen Fibrils; 3.3.2 Staining and Mineralization of the Type-I Collagen Fibrils; 3.3.3 STORM Imaging; 4 Notes; References; Part II: Proteomics Analysis of Collagen Chapter 5: Investigation of Triple-Helix Collagen Hydroxylation by Solid-State NMR Spectroscopy1 Introduction; 1.1 NMR Overview; 1.2 Chemical Shift; 1.3 Solid-State NMR and Magic Angle Spinning; 1.4 Sensitivity of NMR Experiments; 1.4.1 Isotopic Enrichment; 1.4.2 Dynamic Nuclear Polarization in Solid-State NMR; 2 Materials; 2.1 NMR Equipment; 2.2 Solid Biological Samples; 2.3 Stable Radicals for DNP Enhancement; 3 Methods; 3.1 Preparing the Radical Stock Solution for DNP Enhancement; 3.2 Packing the ssNMR Rotor; 3.3 Inserting Rotor into Magnet and Magic Angle Spinning
Series Title: Methods in molecular biology (Clifton, N.J.), v. 1944.
Responsibility: edited by Irit Sagi and Nikolaos A. Afratis.

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