Name: making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry
Uploaded: 2018-05-27T14:00:00
Description: Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

J.J.G. verdikleri destek için Ulusal Bilim Vakfı (DMR-1654405) ve kanser önleme Araştırma Enstitüsü of Texas (CPRIT) (RP170752s) kabul eder.

1. hazırlık
<ol><li>Lysogeny suyu (LB) agar yapmak ve tabak12dökün.</li>
	<li>
bl21(de3) wtQβ kat protein içeren bir pET28 plazmid ile dönüşümü.
	<ol>
<li>E. coli BL21(DE3) yetkili hücrelerde bir buz banyosu çözülme. Hücre microcentrifuge tüp içinde yer 50 μL.</li>
		<li>Plazmid 2 μL bir tüpün içine ekleyin ve hafifçe tüpü hafifçe vur. O zaman 30 dk için buz üzerinde kuluçkaya.</li>
		<li>Isı şok hücreler için 45 s tam olarak 42 ° C'de...

<ol>
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Pouring LB Agar Plates Available from: <a target="_blank" href="https://www.addgene.org/protocols/pouring-lb-agar-plates/">https://www.addgene.org/protocols/pouring-lb-agar-plates/</a> (2016)</li><li>Smith, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Modification,+Bioconjugation,+and+Disulfide+Bridging+Using+Bromomaleimides.">Protein Modification, Bioconjugation, and Disulfide Bridging Using Bromomaleimides.</a> J. Am. Chem. 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Daha küçük protein arıtma için karşılaştırıldığında, bakteriyofaj Qβ arındırıcı bir benzersiz sükroz degrade Santrifüjü adımdır. Kloroform/n-butanol ayıklama adım sonra Qβ daha fazla % 5-40 sükroz degradeler kullanarak saf. Santrifüjü sırasında parçacıklar kendi boyutları göre ayrılır. Daha küçük partiküller daha düşük yoğunluklu bölgede kalırken daha büyük partiküller yüksek yoğunluk bölgesine seyahat. Qβ için geçişin üçüncü alt seyahat ve daha küçük protei...

Nano ölçekli viral capsids kullanarak uygulamaları Biyomedikal araştırma1,2,3' te kapsamını genişletmek hedefliyor heyecan verici bir alan olarak ortaya çıkmıştır. Recombinantly ifade virüs benzeri parçacıklar (VIP) yapısal olarak virüslere karşı elde edilen ama orijinal viral genetik materyal bulaşıcı olmayan proteinaceous nano tanecikleri geliştirmelerde eksik. Yüzey özellikleri genetik programlı ve her kapsid aynı şekilde önce ve sonrasında olanlar için ifade gibi reaktif yan zincirli amino asitlerin atomistic hassasiyetle sayısını ve konumunu öğrenmek mümkündür. Birçok durumda, iç ve dış yüzeyleri pek çok feasibly bioconjugation tepkiler - Kovalent bağlar arasında bir biomolecule ve sentetik reaksiyonlar yoluyla functionalized solvent maruz amino asit kalıntıları sahip. molekül4,5.
Bioconjugation reaksiyonlar ilgi daha farklı işlevleri nispeten basit bir moda var biomolecules yardım. Molekülleri tedavi uyuşturucu6, floresan Etiketler7 ve polimerler8,9 gibi ilgi önceden sentezlenmiş ve onlar VIP'ler yüzeyinde eklenmeden önce ile karakterizedir. Özellikle ortak bir VIP içinde biyomedikal ve biyomalzeme araştırma bakteriyofaj Qβ, recombinantly olarak ifade, 28 nm icosahedral viral kapsid10temel VIP oldu. Son zamanlarda Qβ gözenekleri Haddleton-Baker tepki ile satır azaltılmış disulfides için dibromomaleimide bileşiklerin başarılı konjugasyon11 tebliğ var olsa Qβ en yaygın tepki sitelerinde geniş bir farkla lysines vardır. Reaksiyon gelirleri ile iyi verim ve eşit olarak da önemlisi, olmadan parçacıkların termal kararlılık kaybetme. Aynı zamanda, bu parçacıklar alımını hücrelere izlemek için kullanılan fiil kaynaklı floresans bu reaksiyon oluşturur. Bu çalışmada, Qβ yüzeyine bir parlak sarı fluorophore sonuç Haddleton-Baker tepki ile Polietilen glikol (PEG) konjugasyon göstermektedir. Hepsi hücreleri tarafından alınır gibi bu parçacıklar daha sonra izlenebilir. İletişim kuralı burada araştırmacılar kendi ilkeleri bir çözelti maruz disulfides içeren diğer birçok VIP'ler için uygulanabilir olmasına rağmen proteinaceous nano tanecikleri Qβ dayalı yeni floresan pegile oluşturmak yardımcı olur.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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			<td height="42" style="height:42px;width:216px;">LB Broth (Miller)&#160;</td>
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			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP366-500</td>
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			<td height="42" style="height:42px;width:216px;">Elga PURELAB Flex 3 Water Purification System</td>
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			<td style="width:119px;">BP362-1</td>
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			<td style="width:119px;">P288-500</td>
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			<td style="width:119px;">096-041053</td>
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			<td style="width:119px;">KW-0066-5KG</td>
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			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:71px;">Alfa Aesar</td>
			<td style="width:119px;">32614-M6</td>
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			<td height="42" style="height:42px;width:216px;">1-Butanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">A399-4</td>
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			<td height="84" style="height:84px;width:216px;">SW 28 Ti Rotor, Swinging Bucket, Aluminum</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">342204: SW 28 Ti Rotor/ 342217: Bucket Set</td>
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			<td height="42" style="height:42px;width:216px;">Type 70 Ti Rotor, Fixed Angle, Titanium, 8 x 39 mL,</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">337922</td>
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			<td height="42" style="height:42px;width:216px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">PI23200</td>
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			<td height="84" style="height:84px;width:216px;">TRIS Hydrochloride</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">T60050-1000.0</td>
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			<td height="42" style="height:42px;width:216px;">Tris(2-carboxyethyl)phosphine hydrochloride</td>
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			<td height="42" style="height:42px;width:216px;">Ammonium Persulfate</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP179-100</td>
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			<td height="42" style="height:42px;width:216px;">FV3000 confocal laser scanning microscope</td>
			<td style="width:71px;">Olympus&#160;</td>
			<td>FV3000&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Labnet Revolver Adjustable Rotator&#160;</td>
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			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">3117-0380</td>
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			<td style="width:119px;">4980-2L</td>
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			<td height="42" style="height:42px;width:216px;">Amicon Ultra-4 Centrifugal Filter Units</td>
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			<td style="width:119px;">3117-0380PK</td>
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			<td height="42" style="height:42px;width:216px;">Bottle, with Cap Assembly, Polycarbonate</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;">41121703</td>
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			<td height="42" style="height:42px;width:216px;">Cylinder, Graduated - Polypropylene 250 mL</td>
			<td style="width:71px;">PolyLab</td>
			<td style="width:119px;">80005</td>
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			<td height="42" style="height:42px;width:216px;">533LS-E Series Steam Sterilizers</td>
			<td style="width:71px;">Getinge</td>
			<td style="width:119px;">533LS-E</td>
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			<td height="42" style="height:42px;width:216px;">TrueLine, Cell Culture Plate, Treated, PS, 96 Well, with Lid</td>
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			<td style="width:119px;">D36-313-CS</td>
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			<td height="42" style="height:42px;width:216px;">Falcon 15 mL Conical Centrifuge Tube</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td></td>
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making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry

Virüs gibi parçacıklar (VIP) Biyomedikal son artış ve malzeme araştırma onların kolaylığı biyosentezi, ayrık boyut, genetik programlama ve biodegradability atfedilen. Onlar onların yüzeyine sentetik ligandlar eklemek için bioconjugation reaksiyonlar için son derece uysal, bioconjugation yöntemleri bu sulu d. capsids tarih aralığında göreceli olarak sınırlı iken. Fonksiyonel Biyomalzeme araştırma yönünü kolaylaştırmak için geleneksel olmayan bioconjugation tepkileri dikkate alınmalıdır. Bu protokol için açıklanan tepki dibromomaleimides solvent bazlı bir VIP maruz disülfür bağları bakteriyofaj Qβ yeni işlevsellik tanıtmak için kullanır. Ayrıca, nihai ürün piyasada bulunan filtre seti kullanarak izlenebilir vitro sonda üreten yararı olan flüoresan,.

Dibromomaleimide türevleri ile yoğunlaşma tepki dibromomaleimide anhidrit ve Birincil aminler15arasında sentez. Alternatif olarak, N-methoxycarbonyl aktif 3,4-dibromomaleimide kullanarak bir hafif sentetik yöntemi16 burada methoxypolyethylene glikol (PEG) için verim DB-PEG (Şekil 1) ile tepki tarafından istismar edildi. NMR Bileşik yapı (Şekil 2) tanımlamak için kullan?...

Watch this Scientific Journal Video about Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry at JoVE.com

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Konjugasyon kaynaklı floresan pegile virüs benzeri moleküller tarafından Dibromomaleimide-disülfür Kimya yapma

Burada, fluorescently Qβ VIP üzerinde disulfides dibromomaleimide ile functionalize için bir yordam mevcut. Biz Qβ ifade ve arıtma, molekülleri dibromomaleimide functionalized sentezi ve dibromomaleimide ve Qβ arasında konjugasyon tepki açıklanmaktadır. Elde edilen Sarı floresan konjuge parçacık bir floresan sonda hücrelere olarak kullanılabilir.

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

Research

JoVE Journal

Bioengineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

Tek Parçacıklar için mikroakışkan tabanlı Hidrodinamik Tuzak

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membran Protein katlanması, Termodinamik Floresans Spektroskopisi ile ölçülür.

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Desorpsiyon Elektrosprey İyonizasyon Kütle Spektrometresi ile Biyolojik Dokuların Görüntüleme

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Biyobozunur Polimer Parçacıklarının Lenf İçi Düğüm Enjeksiyonu

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

MicroRNA Niceleme DNA bağlayıcı Boya Kimya ve Damlacık Dijital PCR kullanarak Sirkülasyon

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Bir Ayarlanabilir Nanopore aracılığıyla Nanopartikül Translokasyon Hızları aracılığıyla Zeta Potansiyeli Belirlenmesi: Bir Örnek olarak DNA modifiye Parçacıklar kullanma

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Parçacıkların Mekansal Takip için Çoklanmış Elektronik Algılama ile mikroakışkan Platformu

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Nanoplasmonic optik kafes içinde mikro parçacıkların bindirme

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Site yönettiği immobilizasyon kemik morfogenetik protein 2 katı yüzeyler için tıklayın kimya tarafından

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Mühendislik kanonik olmayan Amino asitler ve seçici basınç eklenmesi tarafından 'Altın' floresan ve kütle spektrometresi ve floresan göre Protein analizi