Name: detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans
Uploaded: 2018-07-30T13:00:00
Description: Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

C. elegans påkjenningen brukes i denne studien var oppnådd fra Caenorhabditis genetikk sentrum, som er støttet av National Institutes of Health - Office for forskning infrastruktur programmer (P40 OD010440). Dette arbeidet ble støttet av NIH: 1R03AG048578-01 Jun Liang, CUNY-CCRG 1501 Jun Liang og PSC-CUNY 66184-00 44 og 67248-00 45 til juni Liang. Vi takker Cathy Savage-Dunn for vennlig deler hennes laboratoriet plass. Alle fluorescens bilder ble samlet på Queens College Core anlegget. Vi takker William J. Rice for hans kommentarer på manuskriptet.

1. løsninger
<ol><li>
NGM plater
	<ol>
<li>I en 2 L Erlenmeyer bolle, Legg 3 g av NaCl, 17 g agar, 2.5 g av pepton og 975 mL av dH2O. dekket munningen av flasken med aluminiumsfolie. Autoclave kolbe for 50 min. avkjøle den i 20-30 min.</li>
		<li>Deretter legger sterilisert løsninger: 1 mL 1 M CaCl2, 1 mL av 5 mg/mL kolesterol i etanol, 1 mL 1 M MgSO4og 25 mL 1 M KPO4 (pH 6.0) bufferen. Swirl løsninger å blande dem godt.</li>
		<li>Bruker en flytende dis...

<ol>
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W., 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=JNK+regulates+lifespan+in+Caenorhabditis+elegans+by+modulating+nuclear+translocation+of+forkhead+transcription+factor/DAF-16.">JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (12), 4494-4499 (2005).</li><li>Gross, A., 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=Caspase+cleaved+BID+targets+mitochondria+and+is+required+for+cytochrome+c+release,+while+BCL-XL+prevents+this+release+but+not+tumor+necrosis+factor-R1/Fas+death.">Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death.</a> The Journal of Biological Chemistry. 274 (2), 1156-1163 (1999).</li><li>Doerflinger, M., Glab, J. A., Puthalakath, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3-only+proteins:+a+20-year+stock-take.">BH3-only proteins: a 20-year stock-take.</a> The FEBS Journal. 282 (6), 1006-1016 (2015).</li><li>Mayorova, T. D., 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=Cells+containing+aragonite+crystals+mediate+responses+to+gravity+in+Trichoplax+adhaerens+(Placozoa),+an+animal+lacking+neurons+and+synapses.">Cells containing aragonite crystals mediate responses to gravity in Trichoplax adhaerens (Placozoa), an animal lacking neurons and synapses.</a> PLoS One. 13 (1), e0190905 (2018).</li><li>Terron-Camero, L. C., Molina-Moya, E., Sanz-Fernandez, M., Sandalio, L. M., Romero-Puertas, M. 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E., Heestand, B. N., Seong, S., Shtessel, L., Ahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+pairing+during+meiosis+triggers+multigenerational+transgene+silencing+in+Caenorhabditis+elegans.">Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (20), E2667-E2676 (2015).</li><li>Schultz, R. D., Bennett, E. E., Ellis, E. A., Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+extracellular+matrix+organization+by+BMP+signaling+in+Caenorhabditis+elegans.">Regulation of extracellular matrix organization by BMP signaling in Caenorhabditis elegans.</a> PLoS One. 9 (7), e101929 (2014).</li><li>Matus, D. Q., 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=Invasive+cell+fate+requires+G1+cell-cycle+arrest+and+histone+deacetylase-mediated+changes+in+gene+expression.">Invasive cell fate requires G1 cell-cycle arrest and histone deacetylase-mediated changes in gene expression.</a> Developmental Cell. 35 (2), 162-174 (2015).</li><li>Reddy, K. C., Villeneuve, A. 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Denne artikkelen presenteres en rask og effektiv metode for å kontrollere protein subcellular endringer fra cytoplasma til kjernen. Protein uttrykket ble vist av en GFP fusion, mens kjernen strukturen ble bekreftet av DAPI flekker (Figur 3). Siden immunostai- C. elegans proteiner er utfordrende, de fleste C. elegans protein subcellular lokaliseringer kjennetegnes ved å merke dem med markør proteiner som GFP, Laz, mCherry og andre18,<sup...

En endring av protein subcellular lokalisering er en vanlig mekanisme svar på interne eller eksterne signaler som varmestress, sult, oksidativt stress, apoptose, protein fosforylering og andre. For eksempel induserer varmestress medlem FOXO DAF-16 kjernefysiske translokasjon1,2, og pro-apoptotisk BCL-2 protein bud translocates til mitokondrier etter mottak død signalering3,4. Det finnes ulike teknikker å oppdage endringene. En kombinasjon av vestlige blotting og biokjemisk isolere subcellular strukturer (f.eks, mitokondrier eller kjerner) kan godt oppnå mål3. Men krever det et spesifikt antistoff mot protein av interesse. Dermed blir et veletablert antistoff nøkkelen til suksess. En alternativ tilnærming er å merke ulike subcellular strukturer eller organeller med ulike indikatorer som grønne fluorescens protein (GFP), røde fluorescens protein (RFP), gul fluorescens protein (YFP) og mCherry, og i mellomtiden gi protein av interesse med andre indikatorer. Deretter observere dem under AC confocal mikroskop å lokalisere mål5,6. Isotopene er et alternativ valg for å merke målet proteiner og deretter oppdage deres subcellular lokalisering7. Denne metoden krever imidlertid riktig trening og håndtering av radioaktivt avfall. Under omstendigheter som mangel på et spesifikt antistoff, fravær av en skikkelig markør eller scarceness utstyr som AC confocal mikroskop må en alternativ tilnærming vurderes. For å identifisere et protein kjernefysiske translokasjon, er det attraktivt bare Merk målet proteiner med en markør og stain kjerner med kjemisk reagens 4', 6-diamidino-2-phenylindole (DAPI) siden dette krever bare vanlig fluorescens mikroskop.
Immunolabeling C. elegans med antistoffer er utfordrende på grunn av lav permeabilitet eggeskall eller collagenous cuticle rundt dyret. I mellomtiden siden C. elegans proteiner er betydelig avvikende fra deres virveldyr orthologs, gir noen kommersielle selskaper C. elegans med bestemte produkter. Det er vanskelig for lite laboratorium generere C. elegans antistoffer for seg selv. Forskere i samfunnet bruker ofte merket proteiner som markører for å demonstrere en protein lokalisering eller genet uttrykk. Denne artikkelen bruker EXL-1::GFP eksempel spore et protein kjernefysiske translokasjon under varme stress8. En integrert translasjonsforskning exl-1::gfp til dyr genomet brukes stabilt uttrykke genet med gfp fusion. Forskning viste at exl-1 er uttrykt i tarmen, kroppen veggen muskler og andre subcellular strukturer8. Denne protokollen demonstrerer hvordan du synkronisere ormer til fjerde (L4) larvestadiet, utføre en varme stress eksperimentet og oppførsel DAPI flekker, etanol fiksering, aceton fiksering og bildebehandling under vanlige fluorescens mikroskop.

<table>
	<tbody>
		<tr>
			<td>DAPI</td>
			<td>Biotium</td>
			<td>40043</td>
			<td>both Hoechst 33258 and Hoechst 33342 also works well</td>
		</tr>
		<tr>
			<td>OP50</td>
			<td>CGC</td>
			<td>OP50</td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
			<td>https://cgc.umn.edu/</td>
		</tr>
		<tr>
			<td>Kim Wipe</td>
			<td>Kimberly-Clark Corporation</td>
			<td></td>
			<td>soft tissue</td>
		</tr>
		<tr>
			<td>Zeiss&#160; AX10</td>
			<td>Zeiss</td>
			<td></td>
			<td>fluorescence microscope</td>
		</tr>
		<tr>
			<td>Axiovision Rel 4.8</td>
			<td>Zeiss</td>
			<td></td>
			<td>microscope software</td>
		</tr>
		<tr>
			<td>AxioCam MR Rev3</td>
			<td>Zeiss</td>
			<td></td>
			<td>digital camera</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent nail polish gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-20 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipettor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-200 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200-1000 &#181;L pipet tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire worm pick</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting protein subcellular localization by green fluorescence protein tagging and 4',6-diamidino-2-phenylindole staining in caenorhabditis elegans

I denne protokollen, en grønn fluorescens protein (GFP) fusion protein og 4', 6-diamidino-2-phenylindole (DAPI) flekker brukes til å spore protein subcellular lokalisering endringer; spesielt en kjernefysisk translokasjon under en varme stress tilstand. Proteiner reagere tilsvarende til eksterne og interne signaler. En vanlig mekanisme er å endre sin subcellular lokalisering. Denne artikkelen beskriver en protokoll for å spore protein lokalisering som ikke krever et antistoff, radioaktivt merking eller AC confocal mikroskop. I denne artikkelen brukes GFP å merke målet protein EXL-1 i C. elegans, medlem av klorid intracellulær kanal proteiner (CLICs) familien, inkludert pattedyr CLIC4. En integrert translasjonsforskning exl-1::gfp transgene linje (med en pådriver og en full genet sekvens) ble opprettet av transformasjon og γ-stråling, og uttrykker stabilt gene og gfp. Nyere forskning viste at ved varmestress, ikke oksidativt stress, EXL-1::GFP akkumuleres i kjernen. Overlappende GFP signalet med både kjerner strukturen og DAPI signaler bekrefter EXL-1 subcellular lokalisering endringene under stress. Denne protokollen presenterer to forskjellige fiksering metoder for DAPI flekk: etanol fiksering og aceton fiksering. DAPI fargeprotokoll presenteres i denne artikkelen er rask og effektiv og bevarer både GFP signalet og protein subcellular lokalisering endringene. Denne metoden krever bare fluorescens mikroskop med Nomarski, et FITC-filter og et DAPI-filter. Det er egnet for en lite laboratorium innstilling, en student forskning, high school student forskning og bioteknologi klasserom.

Klorid intracellulær kanal proteiner (CLIC) er multifunksjonell proteiner som er svært bevart over arter12. Mye forskning viser at CLICs regulere cellular stress, autophagy, apoptose, kreft, angiogenese og macrophage medfødte immunforsvaret i pattedyr system13,14,15,16 ,17. Det er to CLIC homologs i ...

Watch this Scientific Journal Video about Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans at JoVE.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Denne protokollen demonstrerer hvordan å spore et protein kjernefysiske translokasjon under varmestress med en grønn fluorescens protein (GFP) fusion protein som en markør og 4', 6-diamidino-2-phenylindole (DAPI) flekker. DAPI flekker protokollen er rask og beholder GFP og protein subcellular lokalisering signaler.

Oppdage Protein Subcellular lokalisering av Green fluorescens Protein merking og 4', 6-Diamidino-2-phenylindole farging i Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

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Biology

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Grunnleggende<em&gt; Caenorhabditis elegans</em&gt; Metoder: Synkronisering og observasjon

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Tilberedning av Mgm101 rekombinasjon Protein av MBP-baserte Tagging Strategi

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Protein-protein interaksjoner visualisert ved bimolekylær fluorescens complementation i Tobakk Protoplaster og blader

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Grønn Fluorescent Protein-baserte Expression Screening av membran proteiner i<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Kvantifisering av protein uttrykk og samlokalisering Bruke multiplekset Immuno-histokjemisk Farging og multispektrale bildebehandling

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

Kvantitativ lokalisering av et Golgi Protein av Imaging sin sentrum av fluorescens masse

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Metoder for å studere endringer i iboende Protein samling med alder i Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...