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).

Varning: Alla kemi reaktioner bör utföras i ett ventilerat dragskåp och med hjälp av en labbrock, handskar och skyddsglasögon. Reaktionerna bör också genomföras i en kvävemiljö. Etisk uttalande: Våra som deltar i djurförsök utförs i enlighet med de italienska reglerna om skydd av djur som används för försök och andra vetenskapliga ändamål (DM 116.192) och Europeiska ekonomiska gemenskapen föreskrifter (EGT EG L 358/1 1986/12/18 ). OBS: Syntes av [(3 S) -2-oxoazetidin-3-yl] ammoniumacetat beskriv...

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Enzymaktivitet är fint regleras på olika nivåer, inklusive RNA-transkription, proteinsyntes, proteintranslokation, post-translationell modifiering, och protein-proteininteraktion. Ofta enzymuttryck ensam inte redogöra för sin verksamhet. ABPP utvecklades för att studera aktiviteten av proteiner i deras nativa tillstånd. Två funktioner som krävs: en kemisk sond som kovalent binder till det aktiva stället i ett enzym av intresse och en reporter-tagg för att detektera sond-märkt enzym. <p class="jove_conten...

Vanligt använda metoder för att undersöka uttrycksmönster, interaktioner och funktioner av proteiner, inklusive vätskekromatografi-masspektrometri plattformar för hagelgevär analys 1,2, jäst två-hybrid metoder 3,4, och in vitro-analyser, är begränsade i att de är inte bedöma aktiviteten hos proteiner i deras naturliga tillstånd. Aktivitetsbaserad proteinprofilering (ABPP) kan användas för att fylla detta gap. Vid detta tillvägagångssätt sonder små-molekyl med förmåga att kovalent binda till det aktiva stället i ett enzym av intresse är konjugerade till en reportergrupp som möjliggör måldetektering. Med användning av klickkemi (CC), kan reportern integreras i proben eller kan införas efter målet ingrepp har inträffat 5,6. Det senare förfarandet kräver användning av sonderna innehåller lämpliga kemiska grupper, såsom en terminal alkyn eller azid, som kan modifieras med ett antal reporter reagens via bio-ortogonala reaktioner Such som Cu (I) -catalyzed Huisgen [3 + 2] cykloaddition 7-9 eller Staudinger ligering 10,11. Nyligen avslöjas vi föreningen ARN14686 som den första ABP för in vitro och in vivo detektion av cystein hydrolas, NAAA 12. NAAA katalyserar hydrolytiska deaktivering av mättade och enkelomättade FAE, inklusive oleoylethanolamide (OEA) och palmitoyletanolamid (PEA), som är endogena agonister av den antiinflammatoriska nukleär receptor PPAR-alfa 13-15. NAAA huvudsakligen uttryckt i makrofager och andra monocythärledda celler, såväl som i B-lymfocyter 14,16, vilket tyder på en roll i regleringen av det medfödda immunsvaret. Enzymet syntetiseras i den grova endoplasmatiska retiklet i en inaktiv form och aktiveras i sura avdelningarna i cellen genom en autoproteolytisk mekanism 17. Den autoproteolytisk klyvning genererar en ny N-terminal cystein (C131 hos möss och råttor, C126 hos människa), är att nukleofilen ansvarig för FAE hydrolys 18,19. Farmakologisk hämning av NAAA aktivitet ändrar FAE balans syntes / nedbrytning till förmån för ökade cellulära nivåerna av FAE: 16,20,21. Flera β-lakton och β-laktamderivat har visats hämma NAAA aktivitet med hög potens och selektivitet 16,22-26. Dessa inhibitorer fungerar genom S-acylering av den katalytiska cystein 16,27,28. Föreningen ARN14686 designades baserat på den kemiska strukturen hos den systemiskt aktiva, serin-härledda β-laktam NAAA-inhibitor, ARN726 (4-cyclohexylbutyl- N - [(S) -2-oxoazetidin-3-yl] karbamat) 16. Den 4-butyl-cyklohexyl grupp ARN726 ersattes med en C9 mättad alifatisk kedja som bär en terminal alkyn tagg för efterföljande CC konjugering med en azid bärande reporter tagg. Vi valde att konstruera en två-stegs ABP att minimally förändra strukturen av den ursprungliga ställningen och upprätthåller därmed affiniteten av sonden för NAAA. Dessutom undviker införandet av skrymmande taggar, kan en sådan sond vara lämpligare för behandling in vivo än en direkt ABP. ARN14686 hämmar NAAA med hög potens (hNAAA IC 50 = 6 nM rNAAA IC 50 = 13 nM) genom att bilda en kovalent addukt med den katalytiska cystein av enzymet 12. Experiment i levande råttor visade att sonden är selektiv fånga NAAA uttryckt i lungorna. Syra ceramidas, en annan cystein amidas som delar 33-34% identitet med NAAA, identifierades också som en låg affinitet mål när du använder höga sondkoncentrationer (10 um vitro, 10 mg / ml intravenös, iv) 12. Vi har även använt ARN14686 för att studera närvaron av aktiva NAAA i inflammerade råttvävnader efter administration av fullständigt Freunds adjuvans (CFA) 29. Här, vi beskriva ett protokoll för preparatining ARN14686 (Figur 1) och dess tillämpning på utredningen av NAAA aktivering ex vivo. Som ett exempel, beskriver vi ett experimentellt förfarande för att visualisera NAAA i rått tassar efter CFA administrering. I detta experiment, är proteiner som extraherats från tassvävnad efter intravenös injektion av sonden, och ABP-märkta proteomet utsattes för CC med biotin-azid. Biotinylerade prover anrikas med användning av streptavidinpärloma, och proteinblotting utförs. I en annan tillämpning beskriver vi lokalisering av aktiva NAAA med fluorescensmikroskopi i muslungor från prob-behandlade möss. I detta fall är den vävnad sektioneras och sektionerna utsätts för CC för rodamin tillsats. Ett arbetsflöde system visas i figur 2.

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		<col />
		<col />
		<col />
	</colgroup>
	<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

Aktivitetsbaserad proteinprofilering (ABPP) är en metod för identifiering av ett enzym av intresse i en komplex proteomet genom användning av en kemisk sond som riktar enzymets aktiva platser. En reporter tag införs i sonden möjliggör påvisande av det märkta enzymet genom in-gel fluorescens skanning, protein blot, fluorescensmikroskopi, eller vätskekromatografi-masspektrometri. Här beskriver vi framställningen och användningen av föreningen ARN14686, ett klickkemi aktivitetsbaserad sond (CC-ABP) som selektivt känner igen enzymet N -acylethanolamine syra amidas (NAAA). NAAA är en cystein hydrolas som främjar inflammation genom att inaktivera endogena peroxisomproliferator-aktiverad receptor (PPAR) -alfa agonister såsom palmitoyletanolamid (PEA) och oleoylethanolamide (OEA). NAAA syntetiseras som ett inaktivt fullängds proenzym, som aktiveras av autoproteolys i det sura pH-värdet i lysosomen. Lokaliseringsstudier have visat att NAAA huvudsakligen uttryckt i makrofager och andra monocythärledda celler, såväl som i B-lymfocyter. Vi ger exempel på hur ARN14686 kan användas för att detektera och kvantifiera aktiv NAAA ex vivo i gnagare vävnader genom proteinblot och fluorescensmikroskopi.

ARN14686 designades baserat på ställningen av inhibitor ARN726 den NAAA. Den 4-butyl-cyklohexyl grupp ARN726 ersattes med en C9 mättad alifatisk kedja som bär en terminal alkyn tag (figur 1). Alkynen tag infördes för att tillåta användning av ett förfarande i två steg märkning för att lägga till en fluorofor eller en biotin molekylen via CC. Detta särdrag gör ARN14686 ett mycket mångsidigt verktyg för att sondera NAAA in vitro och in vivo.</...

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

Här beskriver vi framställning och användning av en aktivitetsbaserad sond (ARN14686, undec-10-ynyl- N - [(3S) -2-oxoazetidin-3-yl] karbamat) som gör det möjligt att upptäcka och kvantifiera aktiva formen av proinflammatoriska enzym N -acylethanolamine syra amidas (NAAA), både in vitro och ex vivo.

förberedelse och<em&gt; In Vivo</em&gt; Användning av en aktivitetsbaserad Probe för<em&gt; N</em&gt; -acylethanolamine Acid amidas

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 metod för isolering av högrenat RNA från frön för användning i kvantitativ transkriptom analys

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

Framställning av Giant vesiklar inkapslande mikrosfärer genom centrifugering av en vatten-i-olja-emulsion

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

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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

Beredning av Keratin hydrolysat från kyckling fjädrar och dess tillämpning i kosmetika

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

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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

Användning av Alu Element som innehåller Minigenes för att analysera cirkulära RNA

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

Provberedning för probe electrospray jonisering Masspektrometri

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

Beredning av högkvalitativ fermenterad 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

Användning av mikroskala termofores för att mäta protein-lipidinteraktioner

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

Växtprovberedning för mätning av nukleosidan/nukleotidinnehåll med hplc-ms/ms

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