Assays (FRAP और फ्लिप) photobleaching भ्रूण स्टेम कोशिकाओं में रहते हैं chromatin प्रोटीन गतिशीलता उपाय
Summary
हम photobleaching के बाद प्रतिदीप्ति रिकवरी (FRAP) और (फ्लिप) photobleaching के लिए भ्रूण स्टेम कोशिकाओं (ते) में chromatin प्रोटीन की गतिशीलता की निगरानी में प्रतिदीप्ति हानि सहित photobleaching तरीकों का वर्णन. Chromatin प्रोटीन गतिशीलता, जो chromatin plasticity के अध्ययन का मतलब है माना जाता है pluripotent कोशिकाओं में बढ़ाया है.
Abstract
Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area – mediated by the replacement of the bleached molecules with the unbleached molecules – is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein’s mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.
Protocol
Discussion
सबसे उपलब्ध तकनीकों के विपरीत, जो सेल आबादी या तय कोशिकाओं, FRAP प्रयोगों से शुद्ध chromatin शामिल chromatin जीवित कोशिकाओं में प्रोटीन गतिशीलता में बदलाव का पालन करें. हम chromatin प्रोटीन गतिशीलता chromatin plasticity के लिए एक अच्छा…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
हम Meshorer प्रयोगशाला के सदस्यों, खासकर Shai Melcer, आदि Alajem, Edupuganti रघु राम, बड़ी श्री Sailaja, अन्ना Mattout और अल्वा Biran महत्वपूर्ण टिप्पणियों के लिए और एक दैनिक आधार पर मुसीबत शूटिंग photobleaching प्रयोगों के लिए धन्यवाद. EM जोसेफ एच. और बेले आर ब्रौन लाइफ साइंसेज में वरिष्ठ व्याख्याता है और इसराइल विज्ञान फाउंडेशन (943/09 ISF), इसराइल स्वास्थ्य मंत्रालय (6007) यूरोपीय संघ (IRG – 206,872 और 238,176) द्वारा समर्थित है, इसराइल कैंसर रिसर्च फाउंडेशन, हिब्रू विश्वविद्यालय और इसराइल Psychobiology संस्थान के आंतरिक अनुप्रयोगी चिकित्सा अनुदान.
Materials
| Name of the reagent | Company | Catalogue number |
|---|---|---|
| DMEM | Sigma | D5671 |
| Gelatin | Merck | 1.04078 |
| Opti-MEM | Gibco | 31985 |
| TransIT-LT1 | Mirus | MIR2300 |
| 8-well μ-Slides | ibidi | 80826 |
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