Henkenjohann, Sigrun: Highly sensitive fluorescent methods for the detection of enzymes and the determination of their activity by means of specific hydrolases. 2009
Inhalt
- Zusammenfassung
- Abstract
- Table of Contents
- 1 Introduction
- 2 Theoretical Background
- 2.1 Principles of Fluorescence
- 2.2 Fluorescence Spectroscopy
- 2.3 Fluorescence Microscopy
- 2.4 Enzymes
- 2.5 Fluorescence based Enzyme Research
- 3 Materials and Methods
- 3.1 Hardware Devices and Setups
- 3.1.1 Absorption Spectrometer
- 3.1.2 Fluorescence Spectrophotometer
- 3.1.3 Multiplate-Fluorescence Reader
- 3.1.4 High Performance Liquid Chromatograph - HPLC
- 3.1.5 Zeiss Laser Scanning Microscope 710 - LSM
- 3.1.6 Total Internal Reflection Fluorescence Microscope
- 3.2 Samples and their Preparation
- 3.2.1 Enzymes and corresponding Buffers
- 3.2.2 (Fluorescent) Substrates and Modifications
- 3.2.3 CPA Single Molecule-Localization
- 3.2.4 Cell Cultures
- 3.2.5 Chemicals for Immunostaining
- 3.2.6 Labeling Protocols
- 3.3 Measurements and Data Evaluation
- 4 Results and Discussion
- 4.1 Enzyme Investigations with fluorescent Substrates
- 4.1.1 Substrate Characterization
- 4.1.2 Enzyme Kinetics
- 4.1.3 Michaelis-Menten Kinetics
- 4.1.4 Determination of Label Effect to Substrate Acceptance
- 4.1.5 Evaluation of ideal Temperature Settings
- 4.1.6 Single Molecule Enzyme Localization
- 4.1.7 Enzymes in Cells
- 4.2 Enzyme Investigations via Immunostaining
- 5 Conclusion and Outlook
- Bibliography
- Appendix A - Publications
- Appendix B - Protocols
- B.1 Protocol for Preparation of Mowiol-DABCO
- B.2 Original Labeling Protocol
- B.3 Protocol for Examination of labeling Procedure
- B.4 Standard labeling Protocol for triple stained mammalian Cells
- B.5 Labeling Protocol for dSTORM Measurement
- Appendix C - Additional Images