Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software
| Autor: | S.C. Griffiths, Christian Siebold, Luis Alvarez, Jonathan Elegheert, Sergi Padilla-Parra, Rory Nolan |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Brightness Microscope Materials science General Immunology and Microbiology General Chemical Engineering General Neuroscience Confocal Detector Photobleaching General Biochemistry Genetics and Molecular Biology law.invention 03 medical and health sciences 030104 developmental biology law Instrumentation (computer programming) Data pre-processing Spectroscopy Biological system |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|