Name: preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase
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Description: Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

The authors thank the Nikon Imaging Center at Istituto Italiano di Tecnologia, Genova, Italy (NIC@IIT).

Forsiktig: Alle kjemi reaksjoner bør utføres i en ventilert avtrekkshette og med bruk av en laboratoriefrakk, hansker og vernebriller. Reaksjonene må også utføres i et nitrogenmiljø. Etisk uttalelse: Våre prosedyrer som involverer dyr er utført i samsvar med de italienske forskrift om vern av dyr som brukes til eksperimentelle og andre vitenskapelige formål (DM 116 192) og Det europeiske økonomiske fellesskap regelverket (EUT av EF L 358/1 12/18/1986 ). MERK: Syntese av [(3 S) -2-oksoazetidin-3-yl] ammoniu...

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Enzymaktivitet er fint regulert på forskjellige nivåer, inkludert RNA-transkripsjon, proteinsyntese, proteintranslokasjon, post-translasjonell modifikasjon, og protein-protein-interaksjon. Ofte enzymet uttrykk alene ikke høyde for sin aktivitet. ABPP ble utviklet for å studere aktiviteten av proteiner i sin opprinnelige tilstand. To funksjonene er nødvendig: en kjemisk sonde som kovalent binder seg til det aktive sete av et enzym av interesse, og en reporter-tag for å detektere sonde-merket enzym. <p class="jo...

Vanligvis brukes fremgangsmåter for å undersøke uttrykk mønstre, interaksjoner og funksjoner av proteiner, inkludert væskekromatografi-massespektrometri plattformer for hagle analyse 1,2, gjær to-hybrid metoder 3,4, og in vitro-analyser, er begrenset ved at de er ute av stand til å vurdere aktiviteten av proteiner i sin opprinnelige tilstand. Aktivitetsbaserte protein profilering (ABPP) kan brukes til å fylle dette gapet. I denne tilnærmingen sonder små-molekyl er i stand til kovalent binding til det aktive sete av et enzym av interesse er konjugert til en rapportørgruppe som gir mulighet for påvisning av mål. Ved hjelp av klikk kjemi (CC), kan reporteren være integrert inn i sonden eller kan innføres etter mål inngrep har funnet sted 5,6. Den sistnevnte metode krever bruk av prober som inneholder passende kjemiske grupper, for eksempel en terminal alkyn eller azid, som kan modifiseres med en rekke rapportør reagenser via bio-ortogonale reaksjoner such som Cu (I) -catalyzed Huisgen [3 + 2] cykloaddisjon 7-9 eller Staudinger ligering 10,11. Nylig har vi beskrevet forbindelsen ARN14686 som den første ABP for in vitro og in vivo deteksjon av cystein hydrolase, naaa 12. Naaa katalyserer den hydrolytiske deaktivering av mettede og monoumettede Fåes, inkludert oleoylethanolamide (OEA) og palmitoylethanolamide (PEA), som er endogene agonister for den anti-inflammatoriske nukleær reseptor PPAR-alfa 13-15. Naaa uttrykkes hovedsakelig i makrofager og andre monocytt-avledede celler, så vel som i B-lymfocytter 14,16, hvilket antyder en rolle i reguleringen av den medfødte immunrespons. Enzymet blir syntetisert i den grove endoplasmatiske retikulum i en inaktiv form, og aktiveres i sure i cellen ved hjelp av en mekanisme 17 autoproteolytisk. Autoproteolytisk cleavage genererer en ny N-terminale cystein (C131 i mus og rotter, NOK 126 hos mennesker), er at nukleofilen er ansvarlig for hydrolyse FAE 18,19. Farmakologisk hemming av naaa aktivitet endrer FAE syntese / nedbryting av balansen i favør av økt cellulære nivåer av Faes 16,20,21. Flere β-lakton og β-laktam-derivater er blitt vist å hemme aktiviteten naaa med høy potens og selektivitet 16,22-26. Disse inhibitorene virker gjennom S -acylation av den katalytiske cystein 16,27,28. Forbindelsen ARN14686 ble designet basert på den kjemiske strukturen til systemisk aktiv, serin-avledede β-laktam naaa inhibitor, ARN726 (4-cyclohexylbutyl- N - [(S) -2-oksoazetidin-3-yl] karbamat) 16. 4-butyl-cykloheksyl gruppe av ARN726 ble erstattet med en C9 mettet alifatisk kjede som bærer en terminal alkyn kode for etterfølgende CC konjugering med et azid bærende reporter tag. Vi valgte å lage en to-trinns ABP å minimally endre strukturen av den opprinnelige stillaset, og dermed opprettholde affiniteten av sonden for naaa. Dessuten unngår innføring av voluminøse koder, kunne en slik sonde være mer egnet for in vivo-behandling enn en direkte ABP. ARN14686 hemmer naaa med høy potens (hNAAA IC50 = 6 nM, rNAAA IC 50 = 13 nM) ved å danne en kovalent addukt med den katalytiske cystein av enzymet 12. Eksperimenter i levende rotter viste at proben er selektivt i å fange naaa uttrykt i lungene. Acid ceramidase, en annen cystein amidase som deler 33-34% identitet med naaa, ble også identifisert som et lav-affinitet mål ved bruk av høy-probe-konsentrasjoner (10 uM in vitro, 10 mg / ml intravenøs, IV) 12. Vi har også anvendt ARN14686 for å studere nærværet av aktiv naaa i betent vev fra rotte etter administrering av Freunds fullstendige adjuvans (CFA) 29. Her skisserer vi en protokoll for preparatipå av ARN14686 (figur 1) og dens anvendelse i etterforskningen av naaa aktivering ex vivo. Som et eksempel, beskriver vi en eksperimentell prosedyre for å visualisere naaa i rotte potene etter CFA administrering. I dette eksperimentet blir proteinene ekstrahert fra labb vev etter intravenøs injeksjon av sonden, og ABP-merkede proteom- blir underkastet CC med biotin-azid. Biotinylerte prøver er anriket ved hjelp av streptavidin perler, og proteinblottene blir utført. I et annet program, beskriver vi lokalisering av aktive naaa ved fluorescens mikroskopi i muse lungene fra probe-behandlede mus. I dette tilfellet er vevet i snitt og deler er underkastet CC for rhodamin tilsetning. En arbeidsflyt ordningen er illustrert i figur 2.

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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">1,1&#8217;-sulfonyldiimidazole&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">367818</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-dipyridylcarbonate</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">11331</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-Methylbutan</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M32631</td>
			<td>Flamable, toxic,hazardous to the aquatic environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">4-(Dimethylamino)pyridine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">107700</td>
			<td style="width:471px;">Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetic acid</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">695092</td>
			<td style="width:471px;">Flammable, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetonitrile&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34998</td>
			<td style="width:471px;">Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Activated charcoal</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">161551</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ammonium chloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A9434</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Biotin</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ104P4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Fluor 545</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">BCA protein assay kit</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bio-spin columns</td>
			<td style="width:205px;">Biorad</td>
			<td style="width:111px;">732-6204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Biotin</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Blocking buffer</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">b-mercaptoethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M6250</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bovin serum albumine (BSA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bromophenol blue</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bruker Avance III 400</td>
			<td style="width:205px;">Bruker</td>
			<td style="width:111px;"></td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Celite</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">419931</td>
			<td style="width:471px;">Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ceric ammonium nitrate&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">22249</td>
			<td style="width:471px;">Oxidizing, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Chloral hydrate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">C8383</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">CuSO4.5H2O&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">209198</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexadiene</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">125415</td>
			<td style="width:471px;">Flammable, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34855</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dichloromethane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34856</td>
			<td style="width:471px;">Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Diethyl ether</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">296082</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:205px;">Acros Organics</td>
			<td style="width:111px;">348441000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide d6 (DMSO-d6)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">175943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">2860</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethyl acetate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34858</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Glycerol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Irdye 680-LT Streptavidin</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">925-68031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">IRDye680-LT Streptavidin&#160;</td>
			<td style="width:205px;">Licor</td>
			<td style="width:111px;">925-68031</td>
			<td>Briefly centrifuge before use to precipitate protein complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34966</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34860</td>
			<td style="width:471px;">Flammable, Toxic, Health hazard</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">E7750</td>
			<td style="width:471px;">Harmful, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-diisopropylethylamine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">D125806</td>
			<td style="width:471px;">Flammable, Corrosive, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-dimethylformamide</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">227056</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N-Cbz-L-Serine</td>
			<td style="width:205px;">Fluorochem&#160;</td>
			<td style="width:111px;">M03053</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Nikon A1 confocal microscopy</td>
			<td style="width:205px;">Nikon</td>
			<td style="width:111px;"></td>
			<td>Read&#160; the user manual</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">NuPAGE 4-12% Bis-Tris gel</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">NP0335BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Palladium on carbon</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">330108</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">p-anisidine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A88255</td>
			<td style="width:471px;">Toxic, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Paraformaldehyde</td>
			<td style="width:205px;">sigma Aldrich</td>
			<td style="width:111px;">441244</td>
			<td>Toxic, respiratory harmful, corrosive, falmable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol)&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P3265</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">ProLong Gold antifade mountant with DAPI&#160;</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">P36931</td>
			<td>Avoid bubbles formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Protease inhibitor cocktail</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium bicarbonate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">S6014</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium dodecyl sulfate (SDS)&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">L3771</td>
			<td>Toxic, corrosive, falmmable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium hydride&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">452912</td>
			<td style="width:471px;">Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium sulfate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">239313</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Starion FLA-9000 immage scanner</td>
			<td style="width:205px;">FUJIFILM</td>
			<td style="width:111px;"></td>
			<td>Read&#160; the user manual</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Streptavidin agarose</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td>20349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sucrose</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">S7903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tert-butanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">360538</td>
			<td>Toxic, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tetrahydrofuran</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">186562</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Thiourea</td>
			<td style="width:205px;">Acros Organics</td>
			<td style="width:111px;">424542500</td>
			<td>Toxic, warm at 50 &#176;C to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tris</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">RDD008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">C4706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">678937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Triton-x100</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">X100</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-20</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-80</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P1754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ultra turrax IKA T18 basic tissue homogenizer</td>
			<td style="width:205px;">IKA</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Undec-10-yn-1-ol</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">13739</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Urea</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">U5378</td>
			<td>Toxic, warm at 50 &#176;C to dissolve</td>
		</tr>
	</tbody>
</table>

preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase

Aktivitetsbaserte protein profilering (ABPP) er en fremgangsmåte for identifikasjon av et enzym av interesse i en kompleks proteom- ved bruk av en kjemisk sonde som er rettet mot enzymets aktive seter. En reporter kode innføres i sonden gjør det mulig for påvisning av den merkede enzym ved in-gel fluorescens skanning, protein-blot, fluorescens mikroskopi, eller væskekromatografi-massespektrometri. Her beskriver vi utarbeidelse og bruk av det sammensatte ARN14686, et klikk kjemi aktivitetsbaserte probe (CC-ABP) som selektivt gjenkjenner enzymet N -acylethanolamine syre amidase (naaa). Naaa er en cystein hydrolase som fremmer betennelse ved å deaktivere endogene peroksisomproliferatoraktiverende aktivert reseptor (PPAR) alfa agonister som palmitoylethanolamide (PEA) og oleoylethanolamide (OEA). Naaa blir syntetisert som et inaktivt proenzym full-lengde, som aktiveres ved autoproteolysis i det sure pH-verdien i lysosomet. Lokaliserings studier have vist at naaa uttrykkes hovedsakelig i makrofager og andre monocytt-avledede celler, så vel som i B-lymfocytter. Vi gir eksempler på hvordan ARN14686 kan brukes til å oppdage og kvantifisere aktiv naaa ex vivo i gnager vev av protein blot og fluorescens mikroskopi.

ARN14686 er designet basert på skafottet av naaa inhibitor ARN726. 4-butyl-cykloheksyl gruppe av ARN726 ble erstattet med en C9 mettet alifatisk kjede som bærer et terminalt alkyn tag (figur 1). Alkynet koden ble innført for å tillate bruk av en to-trinns merking fremgangsmåte for å legge til en fluorofor eller et biotin-molekyl via CC. Denne funksjonen gjør ARN14686 et meget allsidig verktøy for å sondere naaa in vitro og in vivo. <p class...

Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Her beskriver vi fremstilling og anvendelse av en aktivitetsbasert sonde (ARN14686, undek-10-ynyl- N - [(3 S) -2-oksoazetidin-3-yl] karbamat) som gjør det mulig for påvisning og kvantifisering av aktive form av proinflammatoriske enzymet N -acylethanolamine syre amidase (naaa), både in vitro og ex vivo.

forberedelse og<em&gt; I Vivo</em&gt; Bruk av en aktivitetsbasert Probe for<em&gt; N</em&gt; -acylethanolamine Acid amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Research

JoVE Journal

Biochemistry

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

En effektiv metode for Isolering av høyt renset RNA fra frø for bruk i Quantitative Analysis transcriptome

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

Utarbeidelse av Giant vesikler innkapsle mikrosfære ved sentrifugering av en vann-i-olje emulsjon

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Eksempel forberedelse til masse massespektrometri-baserte identifikasjon av RNA-bindende områder

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Utarbeidelse av Keratin hydrolyse fra kylling fjær og dens anvendelse i kosmetikk

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

An in vivo Østrogenmangel Mouse modell for screening eksogene østrogen behandlinger av kardiovaskulær dysfunksjon etter overgangsalder

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Bruk av Alu Element som inneholder minigenes å analysere sirkulære RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Prøve forberedelse til sonde elektrospray ionisering massespektrometri

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Preparation of High-Quality Fermented Fish Product

Utarbeidelse av høy kvalitet gjæret fisk produkt

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Bruk av mikroskala termoforese for å måle protein-lipid interaksjoner

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Planteprøveforberedelse for nukleoside/nukleotidinnholdsmåling med en HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...