Name: antibody transfection into neurons as a tool to study disease pathogenesis
Uploaded: 2012-09-26T13:00:00
Description: Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

इस काम के अनुसंधान और विकास के कार्यालय, मेडिकल रिसर्च सर्विस, दिग्गजों मामलों के विभाग द्वारा समर्थित है. इस अध्ययन में एक VA मेरिट समीक्षा पुरस्कार (एमसीएल) और विश्वविद्यालय टेनेसी स्वास्थ्य विज्ञान केंद्र मल्टीपल स्केलेरोसिस रिसर्च फंड द्वारा वित्त पोषित किया गया था.

1. एंटीबॉडी लेबलिंग (1 चित्रा) <ol><li> 0.1 एम NaHCO 3 बफर बनाओ: <ul type="circle"><li> 8.4 छ NaHCO 3 </li><li> 29.2 छ NaCl </li><li> 1 लीटर एच 2 हे </li></ul></li><li> बफर इस समाधान (10.6 छ ना 2 3 सीओ, 29.2 ग्राम NaCl, 1 लीटर 2 एच 0) के साथ 8.4 का एक pH लाओ. </li><li> 70 μl विरोधी hnRNPA1 औ?...

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आदेश में विशिष्ट अणु और उनके अभिव्यक्ति के बारे में hypotheses परीक्षण करने के लिए, जीन और अन्य वैक्टर सामान्यतः कोशिकाओं में ट्रांसफ़ेक्ट हैं. इस प्रक्रिया का अनूठा पहलू यह आसानी से लक्ष्य कोशिकाओं में एंट?...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> फ्लोरोसेंट माइक्रोस्कोप AxioObserver A1 </td><td> Zeiss </td><td></td></tr><tr><td> AxioVision 4.2 संस्करण </td><td> Zeiss </td><td></td></tr><tr><td> विरोधी rhnRNPA1 </td><td> Abcam </td><td> Ab4791 </td></tr><tr><td> FITC विरोधी Rigg लेबल </td><td> औज़ बायोसाइंसेज </td><td> AI20100 </td></tr><tr><td> Atto550 एनएचएस एस्टर </td><td> सिग्मा </td><td> 92835 </td></tr><tr><td> अटल बिहारी deliverin </td><td> औज़ बायोसाइंसेज </td><td> AI20100 </td></tr><tr><td> FBS DMEM/F12 10% 1% एंटीबायोटिक्स </td><td> Gibco </td><td> 11320-033 </td></tr><tr><td> पाली-D-Lysine 8 अच्छी तरह से संस्कृति स्लाइड्स </td><td> BD </td><td> 354632 </td></tr><tr><td> बहुउद्देश्यीय अंग को घुमानेवाली पेशी </td><td> वैज्ञानिक Industriतों इंक </td><td> 151 मॉडल </td></tr><tr><td> DAPI बढ़ते मीडिया </td><td> Millipore </td><td> S7113 </td></tr><tr><td> पीबीएस </td><td> Ambion </td><td> 9626 </td></tr><tr><td> अपोहक </td><td> थर्मो फिशर साइंटिफिक </td><td> 66380 </td></tr><tr><td> अल्ट्रा 0.5 Amicon </td><td> Millipore </td><td> UFC501096 </td></tr><tr><td> Paraformaldehyde (4% समाधान) </td><td> इलेक्ट्रॉन माइक्रोस्कोपी विज्ञान </td><td></td></tr><tr><td> नैनो ड्रॉप </td><td> थर्मो फिशर साइंटिफिक </td><td></td></tr></tbody></table>

antibody transfection into neurons as a tool to study disease pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

Watch this Scientific Journal Video about Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis at JoVE.com

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

करने के लिए बातचीत और आणविक एक intracellular वातावरण में एंटीबॉडी की उपस्थिति से संबंधित तंत्र पर प्रभाव की जांच के लिए एक तेजी से दृष्टिकोण वर्णित है. विधि जीना एक लिपिड निर्माण पर आधारित एक गैर सहसंयोजक जटिल गठन का उपयोग कोशिकाओं में एंटीबॉडी के अभिकर्मक शामिल है. तकनीक अमर सेल लाइनों और प्राथमिक कोशिकाओं के लिए अनुकूल है.

रोग रोगजनन का अध्ययन करने के लिए एक उपकरण के रूप में न्यूरॉन्स में एंटीबॉडी अभिकर्मक

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

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