Alzheimer disease [Elektronische Ressource] : identification and characterization of the putative binding partners of amyloid precursor protein (APP) and cell adhesion molecules as biochemical markers / vorgelegt von Elena Strekalova

Linda Guinee

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• Gesundheit

Informations

• Publié par: universitat_hamburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 60
• Poids de l'ouvrage: 2 Mo

Extrait

Alzheimer disease: Identification and characterization of the
putative binding partners of amyloid precursor protein (APP)
and
cell adhesion molecules as biochemical markers


Dissertation
zur Erlangung des Doktorgrades des Fachbereiches Biologie
der Universität Hamburg



vorgelegt von Elena Strekalova

Hamburg, 2003









Name: Elena Strekalova
Titel der Dissertation: Alzheimer disease: Identification and characterization of
putative binding partners of amyloid precursor protein (APP)
and cell adhesion molecules as biochemical markers
Gutachter: Frau Prof. Dr. M. Schachner
Herr Prof. Dr. L. Renwrantz



I. INTRODUCTION.................................................................................................................1
1. ALZHEIMER DISEASE............................................................................................................1
1.1 Amyloid precursor protein............................................................................................2
1.2 Proteolytic processing of APP......................................................................................5
1.2.1 α-Secretase5
1.2.2 β.............................................................................................................6
1.2.3 γ-Secretase7
2. CELL ADHESION MOLECULES OF IMMUNOGLOBULIN SUPERFAMILY.....................................9
2.1 The cell adhesion molecule L1 ...................................................................................10
2.2 The neural cell adhesion molecule (NCAM) ..............................................................13
2.3 Implication of CAMs in neurological disorders .........................................................18
3. INTRODUCTION TO THE THEORY OF COMPLEMENTARY HYDROPATHY................................19
II. AIMS OF THE STUDY ....................................................................................................20
III. MATERIALS ...................................................................................................................22
1. CHEMICALS .......................................................................................................................22
2. SOLUTIONS AND BUFFERS..................................................................................................22
3. BACTERIAL MEDIA.............................................................................................................27
4. BACTERIAL STRAINS AND CELL LINES................................................................................28
5. CELL CULTURE MEDIA .......................................................................................................28
6. MOLECULAR WEIGHT STANDARDS.....................................................................................28
7. PLASMIDS ..........................................................................................................................29
8. ANTIBODIES30
8.1 Primary antibodies .....................................................................................................30
8.2 Secondary antibodies..................................................................................................32
9. PEPTIDES ...........................................................................................................................32
9.1 Antisense peptides32
9.2 Biotinylated peptides33
IV. METHODS .......................................................................................................................34
1. PROTEIN-BIOCHEMICAL METHODS.....................................................................................34
1.1 SDS-polyacrylamide gel electrophoresis....................................................................34
1.2 Western Blot-analysis .................................................................................................34
1.2.1 Electrophoretic transfer .......................................................................................34
1.2.2 Immunological detection of proteins on Nitrocellulose membranes...................35
1.2.3 Immunological detection using enhanced chemiluminescence...........................35
1.3 Coomassie staining of polyacrylamide gels ...............................................................35
1.4 Silver staining of polyacrylamide gels........................................................................35
1.5 Determination of protein concentration (BCA)..........................................................36
1.6 Enzyme-linked immunosorbent assay (ELISA), binding assay...................................36
1.7 Capture ELISA............................................................................................................36
1.8 Preparation of membrane subfractions......................................................................37
1.9 Solubilisation of membrane fractions.........................................................................38
1.10 Isolation of IgG fractions .........................................................................................38
1.11 Affinity chromatography...........................................................................................39
1.12 Sample preparation for protein sequencing .............................................................39
1.13 Purification of rabbit IgG using protein A ...............................................................40
1.14 Immunoprecipitation ................................................................................................40
2. MOLECULAR BIOLOGY.......................................................................................................40
2.1 Bacterial strains .........................................................................................................40

2.1.1 Maintenance of bacterial strains ..........................................................................40
2.1.2 Production of competent bacteria ........................................................................41
2.1.3 Transformation of bacteria ..................................................................................41
2.2 Plasmid isolation of E. coli.........................................................................................41
2.2.1 Plasmid isolation from 3 ml cultures (Minipreps)...............................................41
2.2.2 Plasmi 15 ml-cultures.................................................................41
2.2.3 Plasmi 500 ml-cultures (Maxipreps)..........................................42
2.3 Enzymatic modification of DNA .................................................................................42
2.3.1 Digestion of DNA................................................................................................42
2.3.2 Dephosphorylation of Plasmid-DNA ..................................................................43
2.3.3 Ligation of DNA-fragments ................................................................................43
2.4 DNA Gel-electrophoresis ...........................................................................................43
2.5 Extraction of DNA fragments from agarose gels .......................................................43
2.6 Purification of DNA fragments...................................................................................44
2.7 Determination of DNA concentrations .......................................................................44
2.8 DNA-Sequencing ........................................................................................................44
3. CELL CULTURE ..................................................................................................................45
3.1 CHO cell culture.........................................................................................................45
3.2 Stable transfection of CHO-cells................................................................................45
3.3 Cell culture of stable transfected CHO cells ..............................................................46
3.4 Preparation of dissociated hippocampal cultures......................................................46
4. IMMUNOCYTOCHEMISTRY .................................................................................................47
4.1 Fixation of hippocampal neurons...............................................................................47
4.2 Immunocytochemistry of fixed hippocampal neurons ................................................47
4.3 Co-capping on hippocampal neurons.........................................................................47
4.4 Confocal laser-scanning microscopy48
5. CLINICAL METHODS...........................................................................................................48
5.1 Patients and collection of cerebrospinal fluid............................................................48
6. COMPUTER ANALYSIS........................................................................................................49
6.1 Sequence analysis .......................................................................................................49
6.2 Statistical analysis .................................