Adhesion molecules in two species of echinoderms [Elektronische Ressource] : Marthasterias glacialis and Holothuria forskali / Malgorzata Baranowska

Malgorzata Baranowska

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2008
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 95 Mo

Extrait

Adhesion molecules in two species of echinoderms
Marthasterias glacialis and Holothuria forskali
Dissertation
zur Erlangung des Grades
Doktor der Naturwissenschaften
Am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz
Malgorzata Baranowska
geb. in Jarocin (Polen)
Mainz, 2008Index
Index
1. Introduction .................................................................................................................. 1
1.1. Background to biological adhesion .................................................................... 1
1.2. Review of echinoderm adhesive system............................................................. 2
1.2.1 Tube feet.................................................................................................... 3
1.2.2 Larval adhesive ......................................................................................... 5
1.2.3 Cuvierian tubules....................................................................................... 6
1.2.3.1 Measurement of adhesion of Cuvierian tubules............................ 7
1.3. Comparison with other invertebrates.................................................................. 8
1.3.1 Permanent adhesion................................................................................... 8
1.3.1.1 Mussels.......................................................................................... 9
1.3.1.2 Barnacles ..................................................................................... 10
1.3.2 Temporary adhesion................................................................................ 10
1.3.2.1 Hermans hypothesis .................................................................... 11
1.4 Non secretory adhesive systems in organisms.................................................. 12
1.5 Application of echinoderms bio-adhesives....................................................... 12
1.5.1 Water resistant adhesive.......................................................................... 13
1.5.2 Antifouling strategies .............................................................................. 14
1.6 Overview of studied animals ............................................................................ 14
1.6.1 Asteroidea................................................................................................ 14
1.6.2 Holothuroidea.......................................................................................... 16
2. Aim of the study......................................................................................................... 17
3. Materials…................................................................................................................. 18
3.1 Chemicals ......................................................................................................... 18
3.2 Equipment......................................................................................................... 21
3.3 Antibodies......................................................................................................... 23
3.4 Markers ............................................................................................................. 23
3.5 Kits…................................................................................................................ 23
3.6 Experimental animals ....................................................................................... 24
3.6.1 Starfish .................................................................................................... 24Index
3.6.2 Sea cucumber .......................................................................................... 24
4. Methods……………………………………………………………………………...25
4.1 Methods used to isolate adhesive material ....................................................... 25
4.1.1 Collection of adhesive material ............................................................... 25
4.1.1.1 Starfish ........................................................................................ 25
4.1.1.2 Sea cucumber .............................................................................. 25
4.1.2 Extraction of adhesive material ............................................................... 26
4.1.2.1 Starfish ........................................................................................ 26
4.1.2.2 Sea cucumber .............................................................................. 27
4.1.3 Purification of crude extract for SDS PAGE analysis ............................. 28
®4.1.3.1 PAGE prep Advance Kit ........................................................... 28
4.1.3.2 Ethanol precipitation ................................................................... 28
4.1.3.2 TCA/Acetone precipitation ......................................................... 29
™4.1.3.3 ReadyPrep 2-D Cleanup Kit ..................................................... 30
4.1.4 SDS Polyacrylomide gel electrophoresis (PAGE) .................................. 30
4.1.4.1 Seminative PAGE ....................................................................... 32
4.1.5 Stains for SDS PAGE .............................................................................. 33
4.1.5.1 Gel Code Blue Stain.................................................................... 33
4.1.5.2 Glycoprotein stain ....................................................................... 34
4.1.5.3 Silver stain................................................................................... 34
4.1.5.4 Imperial Stain .............................................................................. 35
4.1.5.5 Sypro Ruby.................................................................................. 36
4.1.6 Antibody production................................................................................ 36
4.1.7 Western Blot ............................................................................................ 36
4.1.8 Neutralisation of antibody ....................................................................... 38
4.1.9 Immunoprecipitation................................................................................ 38
4.1.10 Electro-elution ....................................................................................... 39
4.1.11 Glass Beads............................................................................................ 41
4.1.12 Gel Filtration.......................................................................................... 42
4.1.13 Digestion................................................................................................ 44
4.1.14 HPLC ..................................................................................................... 45Index
4.2 Methods used for characterisation of adhesive material................................... 48
4.2.1 Protein concentration assays ................................................................... 48
4.2.1.1 Bradford Assay............................................................................ 48
4.2.1.2 Lowry Assay ............................................................................... 48
4.2.2 Measurement of adhesion........................................................................ 50
4.2.3 Preparation sections on slides ................................................................. 56
4.2.4 Immunochemistry.................................................................................... 56
4.2.5 Stain Slides.............................................................................................. 57
4.2.5.1 Haematoxylin & Eosin ................................................................ 57
4.2.5.2 Alcian blue, PAS ......................................................................... 58
4.2.5.3 Coomassie Blue........................................................................... 58
4.2.5.4 Alcian blue, Ponceau, Acid fuchsine .......................................... 59
4.2.5.5 Casson’s Trichrome..................................................................... 59
4.2.5.6 Methylene Blue – Azure B.......................................................... 60
4.2.5.7 Haematoxylin & Oil Red O........................................................ .60
4.2.5.8 Toulidyne blue............................................................................. 61
4.2.6 AFM ........................................................................................................ 61
4.2.7 Wyko ....................................................................................................... 63
4.2.8 Transmission electron microscopy.......................................................... 64
5. Results…... ................................................................................................................. 65
5.1 Sea cucumber Holothuria forskali.................................................................... 65
5.1.1 Isolation and identification of the adhesive protein from the
Cuvierian tubules .......................................................................................... ....65
5.1.1.1 Extraction of Cuvierian tubules................................................... 65
5.1.1.2 Identification of adhesive protein................................................ 67
5.1.1.3 Isolation of identified protein...................................................... 68