Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software
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 mean...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Journal of Visualized Experiments
2018
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| _version_ | 1826293610183655424 |
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| author | Nolan, R Alvarez, L Griffiths, S Elegheert, J Siebold, C Padilla-Parra, S |
| author_facet | Nolan, R Alvarez, L Griffiths, S Elegheert, J Siebold, C Padilla-Parra, S |
| author_sort | Nolan, R |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T03:32:47Z |
| format | Journal article |
| id | oxford-uuid:bb4777d4-92db-4d84-b1ba-7cdee061a494 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T03:32:47Z |
| publishDate | 2018 |
| publisher | Journal of Visualized Experiments |
| record_format | dspace |
| spelling | oxford-uuid:bb4777d4-92db-4d84-b1ba-7cdee061a4942022-03-27T05:15:54ZCalibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis softwareJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:bb4777d4-92db-4d84-b1ba-7cdee061a494EnglishSymplectic Elements at OxfordJournal of Visualized Experiments2018Nolan, RAlvarez, LGriffiths, SElegheert, JSiebold, CPadilla-Parra, SNumber 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. |
| spellingShingle | Nolan, R Alvarez, L Griffiths, S Elegheert, J Siebold, C Padilla-Parra, S Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software |
| title | Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software |
| title_full | Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software |
| title_fullStr | Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software |
| title_full_unstemmed | Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software |
| title_short | Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software |
| title_sort | calibration free in vitro quantification of protein homo oligomerization using commercial instrumentation and free open source brightness analysis software |
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