Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Dette arbeidet ble støttet av Korea Institute for treningsplanlegging og -analyse for teknologi i mat og jordbruk skogbruk (IPET) gjennom Agri-Bioindustry teknologi utvikling programmet, finansiert av Landbruksdepartementet, Food and Rural Affairs (MAFRA ) (116159-02-2-WT011) og skolen av biovitenskap og bioteknologi for BK21 pluss, Universitetet.

1. forberedelse av Medium, forbindelser og reagenser
<ol><li>Forberede B16F10 veksten komplett mediet. Supplement Dulbecco endret Eagle's medium (DMEM) med 10% fosterets bovin serum (FBS) og 1% penicillin/streptomycin (PEST).<ol>
<li>For å gjøre 500 mL komplett medium, bland 445 mL av Dulbeccos endret Eagle's medium (DMEM) med 50 mL av FBS og 5 mL av PEST i en 1 L steriliserte glassflaske. Etter miksing forsiktig, filtrere mediet gjennom et 0,2-µm plastflaske-top filter til en ny steriliserte glassflaske og deretter...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+melanogenesis+and+mechanisms+of+skin-lightening+agents+-+existing+and+new+approaches.">The melanogenesis and mechanisms of skin-lightening agents - existing and new approaches.</a> International Journal of Cosmetic Science. 33 (3), 210-221 (2011).</li><li>Passeron, T., Coelho, S. G., Miyamura, Y., Takahashi, K., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+and+in+situ+hybridization+in+the+study+of+human+skin+melanocytes.">Immunohistochemistry and in situ hybridization in the study of human skin melanocytes.</a> Experimental Dermatology. 16 (3), 162-170 (2007).</li><li>Lee, J. 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J., 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=Dual+inhibitions+of+lemon+balm+(Melissa+officinalis)+ethanolic+extract+on+melanogenesis+in+B16-F1+murine+melanocytes:+inhibition+of+tyrosinase+activity+and+its+gene+expression.">Dual inhibitions of lemon balm (Melissa officinalis) ethanolic extract on melanogenesis in B16-F1 murine melanocytes: inhibition of tyrosinase activity and its gene expression.</a> Food Science and Biotechnology. 20 (4), 1051-1059 (2011).</li><li>Jun, H. J., 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=p-Coumaric+acid+inhibition+of+CREB+phosphorylation+reduces+cellular+melanogenesis.">p-Coumaric acid inhibition of CREB phosphorylation reduces cellular melanogenesis.</a> European Food Research and Technology. 235 (6), 1207-1211 (2012).</li><li>Jun, H. J., 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=Dual+inhibition+of+&#947;-oryzanol+on+cellular+melanogenesis:+inhibition+of+tyrosinase+activity+and+reduction+of+melanogenic+gene+expression+by+a+protein+kinase+a-dependent+mechanism.">Dual inhibition of &#947;-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase a-dependent mechanism.</a> Journal of Natural Products. 75 (10), 1706-1711 (2012).</li><li>Choi, Y. 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=Effects+of+the+isoflavone+puerarin+and+its+glycosides+on+melanogenesis+in+B16+melanocytes.">Effects of the isoflavone puerarin and its glycosides on melanogenesis in B16 melanocytes.</a> European Food Research and Technology. 231 (1), 75-83 (2010).</li><li>Cho, B. R., Jun, H. J., Thach, T. T., Wu, C., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaine+reduces+cellular+melanin+content+via+suppression+of+microphthalmia-associated+transcription+factor+in+B16-F1+murine+melanocytes.">Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes.</a> Food Science and Biotechnology. 26 (5), 1391-1397 (2017).</li><li>Overwijk, W. W., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B16+as+a+mouse+model+for+human+melanoma.">B16 as a mouse model for human melanoma.</a> Current Protocols in Immunology. , (2001).</li><li>Virador, V. M., Kobayashi, N., Matsunaga, J., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+protocol+for+assessing+regulators+of+pigmentation.">A standardized protocol for assessing regulators of pigmentation.</a> Analytical Biochemistry. 270 (2), 207-219 (1999).</li><li>D'Mello, S. A. N., Finlay, G. J., Baguley, B. C., Askarian-Amiri, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+melanogenesis.">Signaling pathways in melanogenesis.</a> International Journal of Molecular Sciences. 17 (7), 1144 (2016).</li><li>Hou, L., Panthier, J. J., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+the+neural+crest-derived+melanocyte+lineage:+interactions+between+KIT+and+MITF.">Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF.</a> Development. 127 (24), 5379-5389 (2000).</li><li>Hedley, S. J., Gawkrodger, D. J., Weetman, A. 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Vi presentert protokoller for evaluering av hypopigmentation aktiviteten til test forbindelser med kulturperler melanocytter. Representant resultatene viste hypopigmentation effekten av arbutin, en tyrosinase inhibitor som hemmet tyrosinase aktivitet og cellular melanin innhold. Disse metodene er mye brukt i anti-melanogenic aktivitet forskning. Bruker disse analyser, vi har også identifisert flere bioaktive forbindelser som har melanogenesis hemmende effekt på B16F10 celler i siste tiåret9<sup...

Melanin spiller en avgjørende rolle i fysiologi, patologi og toksikologi flere organer inkludert hud, øyne og hjernen1. Hovedfunksjoner av melanin er Foto-screening og biokjemiske effekter. Melanin absorberer nær infrarød og synlig lys samt ultrafiolett (UV) stråling, med økt absorpsjon priser på kortere bølgelengder av lys; dermed beskytter melanin vev fra skader forårsaket av synlig lys eller UV stråling2. Melanin er en antioksidant og har en affinitet for metaller og andre giftige kjemikalier; Det kan derfor beskytte vev fra oksidativt og kjemiske stress3. Men forårsaker overdreven produksjon av melanin dermatologiske problemer.
Kvantitet og kvalitet av melanin i huden og iris er de viktigste faktorer som bestemmer fargen av iris og hud. Enkeltpersoner kan ha forskjellige huden fargen preferanser; noen favoriserer garvet hud, mens andre favorisere lysere hud farger. Avhengig av disse konsument-profiler, er hypopigmentation kosmetikk utviklet for å tilfredsstille enkelte markeder for favoriserte huden farger4. Følgelig er studier av hypopigmentation og anti-melanogenic viktige både vitenskapelig og praktisk.
Melanogenesis er den komplekse prosessen med melanin biosyntesen gjennom en rekke enzymatisk og spontan kjemiske reaksjoner i melanocytter. En melanocyte er omgitt av ca 36 keratinocytter melanocyttene er melanin syntese fabrikkene som distribuerer sine produkter til nærliggende keratinocytter. I huden transporteres melanin produsert og lagret i melanosomal rommet av melanocytter til nabokommunene keratinocytter i overhuden via dendrites.
L-Tyrosin fungerer som første underlaget for melanogenesis og enzym-tyrosinase gir to påfølgende reaksjoner som konverterer L-Tyrosin til 3,4-dihydroxyphenylalanine (DOPA) og deretter til dopaquinone. Disse reaksjonene er hastighetsbegrensningen trinnet i melanogenesis5,6. Hypopigmentation aktivitet kan derfor først måles ved å vurdere mobilnettet tyrosinase aktivitet direkte. Gjør melanocyte ekstrakter som inneholder tyrosinase er ruges med DOPA og dopaquinone produsert i prøvene kan måles ved spectrophotometry på 475 nm. Verdiene er normalisert av protein konsentrasjoner av prøver og stoffer med hypopigmentation aktivitet resultere i mindre dopaquinone formasjon sammenlignet med kontrollene.
Dernest kan hypopigmentation aktivitet kvantifiseres ved å måle melanin i kulturperler melanocytter direkte. Etter behandling av celler med test materialet, trekkes melanin under alkaliske forhold og melanin innholdet er kvantifisert ved spectrophotometry på 400 nm. En hypopigmentation agent vil resultere i en lavere melanin innhold enn det kontroller7.
Endelig kan hypopigmentation aktiviteten kvantifiseres Fontana-Masson melanin flekker og påfølgende bildeanalyser. Fontana-Masson flekker, melanin granulater redusere ammoniakk-sølv nitrat til en synlig svart metallic tilstand og svarte områder av celler i mikroskop-bildene representerer mengden av melanin.
En hypopigmentation agent gir vanligvis konsekvent og sammenlignbare resultater med disse tre metoder, som bekrefter at aktiviteten av stoffet er gyldig. Alternativt kan det være nyttig å måle uttrykk for viktige gener og proteiner i melanogenesis svar på test stoff som undersøke hypopigmentation aktivitet. Tyrosinase er tyrosinase-relaterte proteiner (TRP-1) og dopachrome tautomerase (TRP-2) viktige enzymer i melanogenesis8. Den transkripsjon faktor mikroftalmi-assosiert transkripsjon faktoren (MITF) er en mester regulator i melanogenesis og måling av genet/protein uttrykk nivåene eller en promoter aktivitet analysen kan også brukes til å vurdere hypopigmentation aktivitet.

<table>
	<tbody>
		<tr>
			<td>3,4-Dihydroxy-l-phenylalanine</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma</td>
			<td>#320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Arbutin</td>
			<td>Fluka</td>
			<td>#10960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Yakuri Pure Chemicals</td>
			<td>#16223</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>American MasterTech</td>
			<td>AHG0226</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Samchun chemical</td>
			<td>H0255</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bio basic Canada Inc.</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T.Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duksan pure chemicals</td>
			<td>#81</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear fast red</td>
			<td>Merck</td>
			<td>100121</td>
			<td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>J.T. Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate pentahydrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#2163</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Bio Basic Canada</td>
			<td>TB0196</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Union Carbide</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosinase from mushroom</td>
			<td>Sigma</td>
			<td>T3824</td>
			<td>25 KU</td>
		</tr>
	</tbody>
</table>

quantification of hypopigmentation activity in vitro

Denne studien presenterer laboratorium metoder for kvantifisering av hypopigmentation aktivitet i vitro. Melanin, store pigment i melanocytter, er syntetisert svar på flere mobilnettet og miljømessige faktorer. Melanin beskytter hudceller fra ultrafiolett skade, men har også Biofysiske og biokjemiske funksjoner. Overdreven produksjon eller akkumulering av melanin i melanocytter føre dermatologiske problemer, for eksempel fregner, mørke flekker, melasma og føflekker. Kontroll av melanogenesis med hypopigmentation agenter er derfor viktig for personer med klinisk eller kosmetisk behov. Melanin er primært syntetisert i melanosomes av melanocytter i en kompleks biokjemiske prosess kalt melanogenesis, som er påvirket av ytre og indre faktorer, som hormoner, betennelse, alder og ultrafiolett lys eksponering. Vi beskriver tre metoder for å bestemme hypopigmentation aktiviteten av kjemikalier eller naturlige stoffer i melanocytter: måling av 1) mobilnettet tyrosinase aktivitet og 2) melanin innhold og 3) flekker og kvantifisere mobilnettet melanin med bilde analyse.
I melanogenesis gir tyrosinase hastighetsbegrensning trinnet som konverterer L-Tyrosin til 3,4-dihydroxyphenylalanine (L-DOPA) og deretter til dopaquinone. Hemming av tyrosinase er derfor en primær hypopigmentation mekanisme. I kulturperler melanocytter, kan tyrosinase aktivitet kvantifiseres ved å legge L-DOPA som et substrat og måle dopaquinone produksjon av spectrophotometry. Melanogenesis kan også måles ved kvantifisere melanin innholdet. Melanin inneholder mobilnettet brøken ekstraheres med NaOH og melanin er kvantifisert spektrofotometrisk. Endelig kan melanin innholdet kvantifiseres av bildeanalyse etter Fontana-Masson farging av melanin. Selv om resultatene av disse in vitro analyser ikke kan alltid gjengis i menneskelig hud, er disse metodene mye brukt i melanogenesis forskning, spesielt som første skritt å identifisere potensielle hypopigmentation aktivitet. Disse metodene kan også brukes til å vurdere melanocyte aktivitet, vekst og differensiering. Konsistente resultater de tre forskjellige metoder sikre gyldigheten av effekten.

Representant resultatene for hypopigmentation aktivitet av arbutin, en anti-melanogenic sammensatte, i B16F10 melanocyttene er vist nedenfor. Figur 1A viser at arbutin betydelig undertrykt mobilnettet tyrosinase aktiviteten sammenlignet med bilen-behandlet kontrollen. Tilsvarende var melanin innholdet i cellene stimulert med arbutin betydelig redusert sammenlignet med kontrollene (figur 1B). Mikroskopiske bilder av celler med Fon...

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Quantification of Hypopigmentation Activity In Vitro

Vi beskriver tre eksperimentelle metoder for å vurdere hypopigmentation aktiviteten av kjemikalier i vitro: kvantifisering av 1) mobilnettet tyrosinase aktivitet og 2) melanin innhold og 3) måling av melanin av mobilnettet melanin flekker og bilde analyse.

Kvantifisering av Hypopigmentation aktivitet i Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Denne metoden kan være nyttig i utviklingen av funksjonell kosmetikk og hudmiddel med anti-melanogene aktiviteter. Det er det å utføre og resultatet kan oppnås raskt. I tillegg kan denne metoden være nyttig for forskere, som ønsker å identifisere eller undersøke effektene eller forbindelsene eller undersøke antimelanogene eller promelanogene aktiviteter.For å måle til Tyrosinase aktivitet begynne med seeding B16-F10 Melanicides på en fem ganger 10 til fjerde celler per brønn av en 24 brønn plate konsentrasjon i komplett medium i 24 timer i en celle kultur inkubator. Neste dag, erstatte supernatant i hver brønn med DMEM supplert med nylaget test sammensatte og inhibitor kontroll eller en negativ kontroll og returnere platen til inkubatoren i ytterligere 72 timer. På slutten av behandlingen skyll brønnene to ganger med 300 mikroliter kalde pbs per brønn og legg platen på is.Deretter legger du til 300 mikroliter lysisbuffer til hver brønn ved hjelp av en celleskraper for å samle inn cellelylatene etter fem minutter. Overfør de resulterende cellepartikkelsuspensjonene til individuelle 1,5 milliliter mikrosentride rør. Og homogenisere lysates tre ganger i to sekunder på 14, 500 rpm på is for å frigjøre intercellulær Tyrosinase.Pellet cellen rusk ved sentrifugering og overføre supernatanter til individuelle 1,5 milliliter sentrifuge rør på is. Tildel 70 mikroliter av hver supernatant til individuelle brønner av en klar 96 brønnpolystyrenmikroplate og tilsett 140 mikroliter tyrosinase substratløsning til hver brønn av supernatant. Rist platen forsiktig for å blande brønninnholdet.Deretter inkubere platen ved 37 grader celsius i to timer. Og mål til tyrosinaseaktivitet på 475 nanometer på en mikroplateleser. For å måle melanininnholdet i cellene etter å ha samlet lysatet av de behandlede cellene som demonstrert.Samle lysates ved sentrifugering og overføre supernatanter til nye rør. Tilsett 300 mikroliter av en normal natriumhydroksid til hver pellet i en time inkubasjon ved 60 grader Celsius. Etterfulgt av sentrifugering av oppløste cellesuspensjoner.Deretter overfører du 200 mikroliter av supernatantene til hver brønn på en 96-brønnplate. Og mål melanininnholdet på en mikroplateleser ved en 400 nanometer bølgelengde. For Fontana-Masson Staining vasker du de behandlede cellene to ganger med 300 mikroliter kalde pbs og fikser cellene med 200 mikroliter på 10% formalin i en time ved fire grader Celsius.Skyll de tykke cellene med destillert vann og tilsett 200 mikroliter på 58 til 68 grader celsius Ammoniakal sølv arbeidsløsning til hver brønn. I en time inkubasjon ved 37 grader Celsius eller til cellene blir gulbrune i fargen. Skyll de beisede cellene med destillert vann etterfulgt av to minutters inkubasjon i 0,1% gullklorid ved romtemperatur.Skyll gullkloridbehandlede celler med destillert vann og tilsett 200 mikroliter natriumtosulfatoppløsning til cellene. Etter to minutter skyll cellene igjen. Og merk cellene med 200 mikroliter kjernekraft raskt rødt per brønn i fem minutter.Vask deretter de beisede cellene med vann fra springen før avbildning under et lett mikroskop. For terskelanalyse åpner du bildene i bilde J og velger bilde, justering og Fargeterskel. For å måle området farget med Fontana Masson sette lysstyrkeparameterlinjen til null og justere lysstyrken til det punktet hvor alle de svarte eller brune beisede cellene er inkludert.Velg Analyser og analyser partikler, og merk av for summeringsboksen i vinduet Analyser partikler, og klikk deretter greit for å få et sammendrag av resultatene og kopier og lim inn dataene i et regneark. Arbutinbehandling undertrykker tyrosinaseaktiviteten betydelig sammenlignet med kontroll av kjøretøybehandlede celler. Tilsvarende melanin innholdet av celler stimulert med Arbutin er betydelig redusert i forhold til kontrollene.Selv om cellene er morfologisk lik mellom behandlingsgrupper, reduserer Arbutin området av svart pigment sammenlignet med de kontrollbehandlede cellene. Denne metoden er nyttig for aktiviteter screening ved hjelp av vårt sammensatte bibliotek. Det er over hundrevis av prøver som skal testes raskt.Videre kan denne metoden også brukes til å undersøke mekanismen som involverer melanogenese. Etter at de bioaktive kandidatene forbindelser har blitt identifisert, for deres vitnesbyrd om studiet av biologiske mekanismer eller kommersielle anvendelser.

quantification-of-hypopigmentation-activity-in-vitro

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Påvisning av pH-avhengig aktivitet av<em&gt; Escherichia coli</em&gt; Anstand HdeB<em&gt; In Vitro</em&gt; og<em&gt; I Vivo</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Kvantifisering av overflod og lading nivåer av overføring RNAs i Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Kartlegging metabolisme: Overvåking laktat Dehydrogenase aktivitet direkte i vev

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

I Vitro analysen å oppdage tRNA-Isopentenyl Transferase aktivitet

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Kalibrering-fri In Vitro kvantifisering av Protein Homo-oligomerization med kommersielle instrumentering og gratis, åpen kildekode lysstyrke analyseprogramvare

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

En fargemetrisk analysen av citrate syntase aktivitet i Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Syntese av en Deuterert standard for kvantifisering av 2-Arachidonoylglycerol i Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Kvantifisering av protein samspill nettverk dynamikk ved hjelp av multiplekset co-Immunutfelling

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Kvantifisering av koenzym A i celler og vev

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

Kvantifisering med høy gjennomstrømning av syntese og distribusjon av mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Forfatter Spotlight: Bildebasert kvantifisering av mitokondriell DNA-syntese og distribusjon  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

High-throughput bildebasert kvantifisering av mitokondriell DNA-syntese og distribusjon

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...