Name: fluorescence anisotropy as a tool to study protein-protein interactions
Uploaded: 2016-10-21T15:00:00
Description: Watch this Scientific Journal Video about Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions at JoVE.com

Authors acknowledge the financial support from CONACyT project numbers 167359 and 177138, and from DGAPA-UNAM project number IN201615.

1. SBDS-फ़्लैश टैग प्रोटीन अभिव्यक्ति और शोधन नोट: anisotropy प्रयोगों के लिए, एक फ्लैश-टैग अनुक्रम Cys-Cys-प्रो-Gly-Cys-Cys मानव पीसीआर द्वारा अनुक्रम कोडन SBDS की सी टर्मिनस को जोड़ा गया है के लिए इसी। इस का निर्माण अभिव्यक?...

<ol>
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प्रोटीन के साथ अधिकांश जैव रासायनिक प्रयोगों न केवल शुद्ध प्रोटीन, लेकिन यह भी उनमें से बड़ी मात्रा में इस्तेमाल तकनीक पर ध्यान दिए बिना की आवश्यकता होती है। इस कारण से, प्रयोगों के इस प्रकार के लिए इस?...

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			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Spectrophotometer</td>
			<td style="width:112px;">Agilent Technologies</td>
			<td style="width:119px;">G6860AA</td>
			<td style="width:225px;">Cary 60 UV-Vis</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">Shaker</td>
			<td style="width:112px;">ThermoFischer Scientific</td>
			<td style="width:119px;">SHKA4000-7</td>
			<td style="width:225px;">MaxQ 4000 Benchtop temperature range Ambient-15&#176; to 60&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Centrifuge</td>
			<td style="width:112px;">ThermoFischer Scientific</td>
			<td>75004271</td>
			<td style="width:225px;">Heraeus Megafuge 16R</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">FPLC</td>
			<td style="width:112px;">Pharmacia Biotech</td>
			<td style="width:119px;">Discontinued</td>
			<td style="width:225px;">FPLC system conductivity UV-MM II monitor P500 pump fraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Spectrofluorometer</td>
			<td style="width:112px;">Olis</td>
			<td style="width:119px;">No applicable</td>
			<td style="width:225px;">Olis DM 45 with Polarization Toolbox</td>
		</tr>
	</tbody>
</table>

fluorescence anisotropy as a tool to study protein-protein interactions

प्रोटीन, प्रोटीन बातचीत एक जीवित जीव के समारोह में एक आवश्यक भूमिका निभाते हैं। एक बार एक बातचीत पहचान की गई है और मान्य यह संरचनात्मक और यंत्रवत स्तर पर यह चिह्नित करने के लिए आवश्यक है। कई जैव रासायनिक और biophysical तरीकों ऐसे प्रयोजन के लिए मौजूद हैं। उनमें से, प्रतिदीप्ति anisotropy एक शक्तिशाली तकनीक विशेष रूप से इस्तेमाल किया जाता है जब एक fluorophore लेबल प्रोटीन की प्रतिदीप्ति तीव्रता प्रोटीन, प्रोटीन बातचीत पर लगातार बना रहता है। इस तकनीक में, एक fluorophore लेबल प्रोटीन एक उपयुक्त तरंग दैर्ध्य है कि चुनिंदा भेजे किरण के साथ उनके रिश्तेदार उन्मुखीकरण के अनुसार fluorophores की एक सबसेट उत्तेजित की खड़ी ध्रुवीकरण प्रकाश के साथ उत्साहित है। इस प्रकार के रूप में जिसके परिणामस्वरूप उत्सर्जन भी एक दिशात्मकता जिसका क्षैतिज और ऊर्ध्वाधर विमानों में संबंधों को परिभाषित करता anisotropy (आर) है: आर = (मैं वी.वी. -मैं VH) / (मैं वी.वी. + 2I VH) है, जहां मैं वी.वी. और मैं <sयूबी&gt; VH क्षैतिज और ऊर्ध्वाधर घटक के प्रतिदीप्ति तीव्रता, क्रमशः रहे हैं। प्रतिदीप्ति anisotropy एक fluorophore, एक fluorophore एक प्रोटीन है, जो प्रोटीन, प्रोटीन बातचीत पर बदल दिया है से जुड़ी अर्थात् स्पष्ट आणविक आकार की बारी-बारी से प्रसार के प्रति संवेदनशील है। वर्तमान पाठ में, प्रोटीन, प्रोटीन बातचीत का अध्ययन करने के लिए एक उपकरण के रूप में प्रतिदीप्ति anisotropy के उपयोग प्रोटीन Shwachman-डायमंड सिंड्रोम (SBDS) में उत्परिवर्तित और बढ़ाव कारक जैसे-1 GTPase (EFL1) के बीच बाध्यकारी संबोधित करने के लिए एक उदाहरण प्रस्तुत किया गया था। पारंपरिक, एक fluorophore के साथ एक प्रोटीन की लेबलिंग thiol समूह (सिस्टीन) पर या प्रोटीन के अमीनो समूह (एन टर्मिनल अमाइन या लाइसिन) में किया जाता है। हालांकि, SBDS कई cysteines और lysines है कि यह साइट का निर्देश दिया लेबलिंग की अनुमति नहीं दी पास। एक वैकल्पिक तकनीक, डाई 4 &#39;के रूप में, 5&#39;-बीआईएस (1,3,2 dithioarsolan-2-YL) fluorescein विशेष रूप से एक tetracysteine ​​आकृति, Cys- लेबल करने के लिए इस्तेमाल किया गया थाCys-प्रो-Gly-Cys-Cys, आनुवंशिक रूप से पुनः संयोजक SBDS प्रोटीन की सी टर्मिनस में इंजीनियर। प्रयोगात्मक डेटा की फिटिंग इन प्रोटीनों के बीच बंधन मोड पर मात्रात्मक और यंत्रवत जानकारी प्रदान की।

किसी भी anisotropy प्रयोग करने के बाद से यह प्रजाति का एक मिश्रण से मनाया anisotropy 5 समीकरण का प्रतिनिधित्व करती है fluorophore के प्रतिदीप्ति तीव्रता में बड़े परिवर्तन बाहर शासन करने के लिए महत्वपूर्ण है: <p...

Watch this Scientific Journal Video about Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions at JoVE.com

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

प्रोटीन बातचीत एक सेल के समारोह के दिल में हैं। Calorimetric और स्पेक्ट्रोस्कोपी तकनीक आमतौर पर उन्हें चिह्नित करने के लिए इस्तेमाल कर रहे हैं। यहाँ हम प्रोटीन Shwachman-डायमंड सिंड्रोम (SBDS) में उत्परिवर्तित और बढ़ाव कारक की तरह 1 GTPase (EFL1) के बीच बातचीत का अध्ययन करने के लिए एक उपकरण के रूप में प्रतिदीप्ति anisotropy का वर्णन है।

एक उपकरण प्रोटीन, प्रोटीन बातचीत का अध्ययन करने के रूप में प्रतिदीप्ति एनिसोट्रॉपिक

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

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