जी प्रोटीन रिसेप्टर युग्मित spectrally हल दो photon माइक्रोस्कोपी का उपयोग सहभागिता के vivo मात्रा में
Summary
एक spectrally हल दो photon माइक्रोस्कोपी इमेजिंग प्रणाली काम करके, Forster अनुनाद ऊर्जा (झल्लाहट) स्थानांतरण क्षमता के पिक्सेल स्तर के नक्शे झिल्ली रिसेप्टर्स होमोसेक्सुअल oligomeric परिसरों फार्म धारणा व्यक्त की कोशिकाओं के लिए प्राप्त कर रहे हैं. दक्षता नक्शे झल्लाहट, हम अध्ययन के तहत oligomer जटिल के बारे में stoichiometric जानकारी का अनुमान करने में सक्षम हैं.
Abstract
The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. Förster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 α-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.
Protocol
Discussion
इस प्रस्तुति में, हम कैसे और vivo में एक प्रोटीन oligomer जटिल के बारे में जानकारी संरचनात्मक आकार निर्धारित करने के लिए सचित्र. जबकि प्रस्तुत डाटा एक विशिष्ट झिल्ली (यानी Ste2p) रिसेप्टर खमीर कोशिकाओं में व्य?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
यह काम UW-मिलवॉकी अनुसंधान विकास पहल, जैव चिकित्सा और स्वास्थ्य टेक्नोलॉजीज के लिए विस्कॉन्सिन संस्थान, और ब्राडली फाउंडेशन द्वारा समर्थित किया गया था.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Peptone | Fisher Scientific | BP1420 | ||
| Yeast Extract | Fisher Scientific | BP1422 | ||
| Polyethlyene Glycol 4000 | Hampton Research | HR2-605 | ||
| Yeast Nitrogen Base w/o (NH4)2SO4 and amino acids | Fisher Scientific | DF0335-15-9 | ||
| Yeast Synthetic Drop-out Medium Supplements | Sigma Aldrich | Y2001 | ||
| D-Glucose | Fisher Scientific | D16-1 | ||
| Agar | Fisher Scientific | S70210 | ||
| Ammonium Sulfate | Fisher Scientific | A702-500 | ||
| Potassium Chloride | Acros Organics | 424090010 | ||
| Leucine | Fisher Scientific | BP385 | ||
| Histidine | Fisher Scientific | BP382 | ||
| Plan Achromat Infinity Corrected 100x Oil Immersion Objective NA=1.43 | Nikon | |||
| Spectrally resolved two photon microscope |
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