Name: covalent binding of bmp-2 on surfaces using a self-assembled monolayer approach
Uploaded: 2013-08-26T13:00:00
Description: Watch this Scientific Journal Video about Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach at JoVE.com

Vi takker Prof. JP Spatz (Institut for Biofysisk kemi, University of Heidelberg og Institut for nye materialer og Biosystems, Max Planck Instituttet for Intelligent Systems, Stuttgart) for hans venlige støtte. Den finansielle støtte fra Max-Planck-Gesellschaft og Deutsche Forschungsgemeinschaft (DFG SFB/TR79 til ØK-A.), Er også i høj grad anerkendt.

1.. Syntese af 11-Mercaptoundecanoyl-N-hydroxysuccinimidester (MU-NHS) <ol><li> Tilsæt dråbevis en opløsning af 500 mg N-hydroxysuccinimid og 30 mg 4 - (dimethylamino) pyridin i 10 ml acetone (pa) til 1 g 11-mercaptoundecanoic syre i 40 ml dichlormethan (pa) ved stuetemperatur (RT). </li><li> Reaktionen til 0 ° C, afkøles og tilsættes dråbevis 1,1 g N, N &#39;-dicyclohexylcarbodiimid i 10 ml dichlormethan (under nitrogenatmosfære). Reaktionen ved lav temperatur i 1 time Hold og deref...

<ol>
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I denne protokol beskriver vi forberedelse af overflader funktionaliserede med bioaktive rhBMP-2. Denne fremgangsmåde består af to trin: 1) den indledende dannelsen af ​​en selvsamlende monolag (SAM) i en bifunktionel linker på guld overflade, 2) kovalent immobilisering af rhBMP-2 protein. I tidligere arbejde har vi valideret effektiv binding af den bifunktionelle linker og vækstfaktoren, og viste, at overflade-immobiliserede rhBMP-2 bevarer sin biologiske aktivitet 19. Bioaktiviteten af vækstfaktore...

Knoglemorfogenetisk protein 2 (BMP-2) er et medlem af den transformerende vækstfaktor (TGF-β)-familien og fungerer som inducer af de novo knogledannelse såvel som regulator af adskillige væv under fosterudviklingen og voksen homeostase 1-3. Hver monomer af det biologisk aktive homodimere BMP-2-protein indeholder et &quot;cysteinknude&quot; motiv, som er stærkt konserveret i alle BMP&#39;er 4. Seks af de syv cysteinrester danner intramolekylære disulfidbindinger, som stabiliserer hver monomer, mens den syvende cystein er involveret i dimerisation, danner en intermolekylær binding mellem de to monomerer 5,6. Dette stærkt konserverede cysteinknude definerer den tredimensionale struktur af BMP-2-protein og bestemmer dets unikke egenskaber, såsom modstandsdygtighed over for varme, denatureringsmidler og sur pH 7-9. BMP-2 binder til serin / threonin kinase transmembrane receptorer, for derved at inducere signaltransduktion <sup&gt; 10-12. Afhængig af formen af ​​receptoroligomerisering der forskellige signalveje aktiveret: a Smad-uafhængig signaleringskaskade fører til alkalisk fosfatase induktion via p38 signalering, mens en Smad-afhængige vej aktiveres af receptorfosforylering resultater i Smad komplekse nukleare translokation og aktivering af transskription af specifikke målgener, såsom inhibitor af differentiering (Id) 12-14. I knogler, BMP-2 inducerer differentiering af mesenkymale stamceller til osteoblaster og dermed stimulere den helbredende og de ​​novo dannelsen af knogle. I øjeblikket rekombinant udtrykt BMP-2 anvendes klinisk til at øge helingen af ​​brækkede sites. En almindelig strategi i knoglevæv teknik er brugen af ​​injicerbare vækstfaktorer, hvilket er mindre invasiv sammenlignet med lokale afgivelsessystemer. Imidlertid har in vivo-undersøgelser og kliniske anvendelser vist, at den korte biologiske halveringstid, uspecifik localt andet lige og hurtig lokal clearance af BMP-2, kan føre til flere lokale, ektopiske og systemiske problemer 15. Derfor, for at opnå en effektiv præsentation, klemning eller immobilisering af BMP-2 inden for eller på materialer er nødvendigt for dens lokale og vedvarende levering på target site. Vedvarende levering kan opnås med ikke-kovalente tilbageholdelse tilgange, såsom fysisk entrapment, adsorption eller ion kompleksdannelse 16. Imidlertid er det kendt, at uspecifik adsorption af proteiner til overflader kan resulterer i denaturering af molekylerne 17. Til den covalente binding af vækstfaktorer har forskellige typer af understøtninger blevet udviklet i løbet af det sidste årti. Brugen af ​​bifunktionelle forbinder molekyler, der er målrettet amino-eller carboxylgrupper af proteinet for eksempel, er en type af tilgang, der ikke nødvendigvis kræver protein modifikation at nå sit immobilisering. I virkeligheden, mens protein modifikation har den fordel, at kontrollere protein orientering,indførelsen af kunstige domæner, peptid tags og stedspecifikke kæder kan ændre den biologiske aktivitet af vækstfaktorer 17. Således at omgå denaturering på grund af interaktion med det understøttende materiale overflader kan funktionaliseres på forhånd, for eksempel med en selv-samlet monolag (SAM) af et linkermolekyle, efterfulgt af kobling af den ønskede faktor 18. Vi har brugt en SAM tilgang til kovalent immobilisere BMP-2 på en overflade ved at målrette sine frie aminrester og har vist, at det immobiliserede protein bevarer både den korte og langsigtede biologiske aktivitet 19. Denne protokol giver en enkel og effektiv måde at levere BMP-2 til celler til in vitro-undersøgelser på de mekanismer, der opstår ved cellemembranen og regulerer intracellulær signalering ansvarlig for osteogenisk signalering.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(dimethylamino)pyridin</td>
			<td>Sigma-Aldrich</td>
			<td>522805</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>AppliChem</td>
			<td>A2282</td>
			<td></td>
		</tr>
		<tr>
			<td>11-mercaptoundecanoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>674427</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichlormethane</td>
			<td>Merck</td>
			<td>106050</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;-dicyclohexylcarbodiimide</td>
			<td>Sigma-Aldrich</td>
			<td>D80002</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum benzene</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Carl Roth</td>
			<td>M 875</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylacetate</td>
			<td>AppliChem</td>
			<td>A3550</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>4627</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Carl Roth</td>
			<td>T921</td>
			<td></td>
		</tr>
		<tr>
			<td>rhBMP-2</td>
			<td>R&#38;D Systems</td>
			<td>355-BM</td>
			<td>Carrier-free; expressed in E.coli</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>PAA</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>HN00.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(dimethyl siloxane) (PDMS)</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rhBMP-2</td>
			<td>Sigma</td>
			<td>B9553</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG-HRP</td>
			<td>Santa Cruz</td>
			<td>sc-2005</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ampliflu Red assay</td>
			<td>Sigma</td>
			<td>90101</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) (1x), liquid</td>
			<td>Gibco</td>
			<td>41966</td>
			<td>High glucose</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F7524</td>
			<td>Sterile filtered, cell culture tested</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05% (1x) with EDTA 4Na</td>
			<td>Gibco</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine (0.1 M)</td>
			<td>Riedel-de Ha&#235;n</td>
			<td>33226</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630 (1%)</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>Lysis buffer (ALP assay)19</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)(1 mM)</td>
			<td>Carl Roth</td>
			<td>HNO3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc chloride (ZnCl2) (1 mM)</td>
			<td>Carl Roth</td>
			<td>3533.1</td>
			<td></td>
		</tr>
		<tr>
			<td>p-nitrophenylphosphate (pNPP)</td>
			<td>Sigma</td>
			<td>S0942</td>
			<td>Phosphatase substrate</td>
		</tr>
		<tr>
			<td>Anti-mysin heavy chain (MHC)</td>
			<td>Developmental Studies Hybridoma Bank, University of Iowa</td>
			<td>MF20</td>
			<td>Monoclonal antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Ultrsonic bath (Sonorex Super RK 102H), Frequency 35 kHz</td>
			<td>BANDELIN electronic GmbH &#38; Co. KG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MED 020 Sputtercoating system</td>
			<td>BAL-TEC AG</td>
			<td></td>
			<td>Coating conditions 
			Cr: 120 mA, 1.3 x 10-2 mbar, 30 sec 
			Au: 60 mA, 5.0 x 10-2 mbar, 45 sec</td>
		</tr>
		<tr>
			<td>Tecan Infinite M200 Plate reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

covalent binding of bmp-2 on surfaces using a self-assembled monolayer approach

Knoglemorfogenetisk protein 2 (BMP-2) er en vækstfaktor, indlejret i den ekstracellulære matrix af knoglevæv. BMP-2 virker som udløser af mesenkymal celledifferentiering til osteoblaster og dermed stimulere heling og de ​​novo knogledannelse. Den kliniske anvendelse af rekombinant human BMP-2 (rhBMP-2), sammenholdt med stilladser har rejst de seneste kontroverser, baseret på tilstanden af ​​præsentation og det beløb, der skal leveres. Protokollen præsenteres her giver en enkel og effektiv måde at levere BMP-2 for in vitro-undersøgelser på celler. Vi beskriver, hvordan der dannes en selv-samlet monolag bestående af en heterobifunktionel linker, og vise efterfølgende binding trin for at opnå kovalent immobilisering af rhBMP-2. Med denne fremgangsmåde er det muligt at opnå en holdbar præsentation af BMP-2, samtidig med at den biologiske aktivitet af proteinet. Faktisk overfladen immobilisering af BMP-2 giver målrettede undersøgelser ved at forhindre uspecifikke annoncerorption og samtidig reducere mængden af ​​vækstfaktor, og især, at hindre ukontrolleret udslip fra overfladen. Både på kort og lang sigt signaleringsbegivenheder udløst af BMP-2 finder sted, når cellerne udsættes for overflader præsenterer kovalent immobiliseret rhBMP-2, hvilket gør denne metode egnet til in vitro-undersøgelser på celle respons på BMP-2 stimulation.

I vores setup, var guld valgt som hjælpestof, da det giver en biologisk uspecifik men kemisk tunable system. Endvidere kan anvendelsen af ​​selvsamlende monolag indebærer mange fordele: SAM spontant adsorberer via deres &quot;hoved-grupper&quot; på metaller og danner monolag med få fejl, mens deres funktionelle endelige grupper kan modificeres yderligere. Således giver de en platform for at skræddersy egenskaberne for grænsefladen i et kontrolleret endnu meget fleksible måde 20. <p class="jove...

Watch this Scientific Journal Video about Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach at JoVE.com

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Vi beskriver en metode til opstilling af en effektiv immobilisering af BMP-2 på overflader. Vores tilgang er baseret på dannelsen af ​​en selvstændig samlet monolag for at opnå den kovalente binding af BMP-2 via dets frie aminrester. Denne metode er et nyttigt værktøj til at studere signalering på cellemembranen.

Kovalent binding af BMP-2 på overflader med en selv-samlet monolag Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

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Organic Chemistry

Chemistry

Unit 1

Covalent Bonding and Structure

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Dependence of Laser-induced Breakdown Spectroscopy Results on Pulse Energies and Timing Parameters Using Soil Simulants

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Dynamic Electrochemical Measurement of Chloride Ions

Dynamisk Elektrokemisk Måling af chloridioner

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

Facile Udarbejdelse af internt Self-samlet Lipid Partikler Stabiliseret af kulstofnanorør

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Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Microfluidic-based Synthesis of Covalent Organic Frameworks (COFs): A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface

Microfluidic-based Synthesis of Covalent Organic Frameworks COFs: A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface

Mikrofluidbaseret syntese af kovalente organiske rammer (COF&#39;er): Et værktøj til kontinuerlig produktion af COF-fibre og direkte tryk på en overflade

Covalent Organic Frameworks (COFs) are a class of porous covalent materials which are frequently synthesized as unprocessable crystalline powders. The ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

Lys-drevet molekylære motorer på overflader for enkelt molekylær billeddannelse

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Ion Mobility-massespektrometri teknikker til bestemmelse af strukturen og mekanismerne for metal Iongenkendelse og Redoxaktivitet af metal bindende oligopeptider

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

Syntese af informationsbærende Peptoids og deres sekvens-instrueret Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...