Бимолекулярные флуоресценции комплементации
Summary
Субклеточные локализации белков играет важную роль в определении пространственно-временной регуляции клеточной сигнализации. Здесь мы описываем бимолекулярной флуоресценции дополнения (BiFC) как прямой метод мониторинга пространственных взаимодействий белков в клетке.
Abstract
Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.
Protocol
Discussion
BiFC является превосходным методом для визуализации белка: белковых взаимодействий в целых клеток и субклеточных определения локализации этих комплексов. Преимущества BiFC в том, что только взаимодействующие флуоресцентные белки, преходящие взаимодействия стабилизировалась, и пост-обр…
Disclosures
The authors have nothing to disclose.
Acknowledgements
ITSN, PI3K-C2β и управляющие векторы, используемые в настоящем протоколе можно получить у авторов по запросу, в некоммерческих целях. Авторы хотели бы выразить признательность д-р Чан-Дэн Ху за любезное предоставление консультаций и реагенты, используемые в создании BiFC протокол в лаборатории О'Брайен. KAW было поддержано финансирования из Фонда Джерома Лежен. Работа в О'Брайен лаборатории поддержана грантами NIH (HL090651), DOD (PR080428), Фонд святого Baldrick, и Фонд Иероним Лежен.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DMEM | Cellgro | 10-013 | ||
| Fetal bovine serum | Cellgro | 35-011-CV | ||
| Glass Bottom Microwell dishes | Matek | P35G-1.5-14C | ||
| 6-well dishes | Falcon | 35-3846 | ||
| Lipofectamine | Invitrogen | 18324020 | ||
| PBS | Cellgro | 21-031-CV | ||
| Paraformaldehyde | Sigma | P6148 | ||
| Confocal Microscope | Zeiss | LSM510 META |
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