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 stammer anvendes i denne undersøgelse blev indhentet fra byens Caenorhabditis genetik, som støttes af National Institutes of Health - Office for forskning infrastruktur programmer (P40 OD010440). Dette arbejde blev støttet af NIH: 1R03AG048578-01 Jun Liang, CUNY-CCRG 1501 Jun Liang og PSC-CUNY 66184-00 44 og 67248-00 45 til Jun Liang. Vi takker Cathy vild-Dunn for venligst deler hendes laboratorium plads. Alle fluorescens billeder blev indsamlet på Queens College Core facilitet. Vi takke William J. Rice for hans kommentarer til manuskriptet.

1. løsninger
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NGM plader
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<li>I en 2 L Erlenmeyerkolbe, tilsættes 3 g NaCl, 17 g agar, 2,5 g af pepton og 975 mL dH2O. dækker mundingen af kolben med aluminiumsfolie. Autoklave kolbe i 50 min. afkøles det for 20-30 min.</li>
		<li>Derefter tilføje steriliseret løsninger: 1 mL 1 M CaCl2, 1 mL af 5 mg/mL kolesterol i ethanol, 1 mL 1 M MgSO4og 25 mL 1 M KPO4 (pH 6.0) buffer. Swirl løsninger for at bland dem godt.</li>
		<li>Ved hjælp af en f...

<ol>
	<li>Essers, M. 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=FOXO+transcription+factor+activation+by+oxidative+stress+mediated+by+the+small+GTPase+Ral+and+JNK.">FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK.</a> The EMBO Journal. 23 (24), 4802-4812 (2004).</li><li>Oh, S. 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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S., Malik, M., Shukla, A., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4,+skin+homeostasis+and+cutaneous+cancer:+surprising+connections.">CLIC4, skin homeostasis and cutaneous cancer: surprising connections.</a> Molecular Carcinogenesis. 46 (8), 599-604 (2007).</li><li>Yao, Q., Qu, X., Yang, Q., Wei, M., Kong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4+mediates+TGF-beta1-induced+fibroblast-to-myofibroblast+transdifferentiation+in+ovarian+cancer.">CLIC4 mediates TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer.</a> Oncology Reports. 22 (3), 541-548 (2009).</li><li>Ronnov-Jessen, L., Villadsen, R., Edwards, J. C., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+a+chloride+intracellular+channel+gene,+CLIC4,+in+transforming+growth+factor-beta1-mediated+conversion+of+fibroblasts+to+myofibroblasts.">Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1-mediated conversion of fibroblasts to myofibroblasts.</a> The American Journal of Pathology. 161 (2), 471-480 (2002).</li><li>He, G., 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=Role+of+CLIC4+in+the+host+innate+responses+to+bacterial+lipopolysaccharide.">Role of CLIC4 in the host innate responses to bacterial lipopolysaccharide.</a> European Journal of Immunology. 41 (5), 1221-1230 (2011).</li><li>Zhong, 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=Inhibition+of+CLIC4+enhances+autophagy+and+triggers+mitochondrial+and+ER+stress-induced+apoptosis+in+human+glioma+U251+cells+under+starvation.">Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation.</a> PLoS One. 7 (6), e39378 (2012).</li><li>Liang, 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=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130 (26), 6453-6464 (2003).</li><li>Leopold, L. 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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J., Shen, Z. Z., Cunha, A., Emmons, S. W., Leroi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+polyploidization+and+cellular+proliferation+drive+body+size+evolution+in+nematodes.">Somatic polyploidization and cellular proliferation drive body size evolution in nematodes.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (10), 5285-5290 (2000).</li><li>Nagamatsu, Y., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+the+control+of+body+size+by+a+G-kinase+and+a+downstream+TGFbeta+signal+pathway+in+Caenorhabditis+elegans.">Mechanisms for the control of body size by a G-kinase and a downstream TGFbeta signal pathway in Caenorhabditis elegans.</a> Genes to Cells. 9 (1), 39-47 (2004).</li></ol>

I denne artikel præsenteres en hurtig og effektiv metode til at kontrollere protein subcellulært ændringer fra cytoplasmaet til kernen. Protein udtryk var vist af en normal god landbrugspraksis fusion, mens kernen struktur blev bekræftet af DAPI farvning (figur 3). Da immunfarvning C. elegans proteiner er udfordrende, de fleste C. elegans protein subcellulært lokaliseringer er karakteriseret ved at kode dem med markør proteiner som normal god landbrugspraksis, Laz, mC...

En ændring af protein subcellulært lokalisering er en fælles mekanisme som reaktion på interne eller eksterne signaler som varmestress, sult, oxidativ stress, apoptose, protein fosforylering, og andre. For eksempel, inducerer varmestress FOXO medlem DAF-16 nukleare translokation1,2, og den pro-apoptotiske BCL-2 protein bud translocates til mitokondrier ved modtagende død signalering3,4. Der findes forskellige teknikker til at opdage disse ændringer. En kombination af western blotting og biokemisk isolere subcellulært strukturer (fx, mitokondrier eller kerner) kunne godt nå mål3. Det kræver dog et specifikt antistof mod protein af interesse. Således, en veletableret antistof bliver nøglen til succes. En alternativ fremgangsmåde er at mærke forskellige subcellulært strukturer eller organeller med forskellige markører som grønne fluorescens protein (NGL), røde fluorescens protein (RFP), gul fluorescens protein (YFP) og mCherry, og i mellemtiden label protein af interesse med andre markører. Derefter skal overholde dem under en Konfokal mikroskop til at lokalisere mål5,6. Radioaktive isotoper er en alternativ valg for mærkning target proteiner og derefter opdage deres subcellulært lokalisering7. Denne metode kræver imidlertid korrekt træning og håndtering af radioaktivt affald. Under omstændigheder som manglen på en bestemt antistof, mangel på en ordentlig markør eller sjældenhed af udstyr såsom en Konfokal mikroskop skal en alternativ tilgang overvejes. For at identificere et protein nukleare translokation, er det attraktivt at kun mærke mål proteiner med en markør og at pletten kerner med kemisk reagens 4', 6-diamidino-2-phenylindole (DAPI) da dette kun kræver en regelmæssig fluorescens mikroskop.
Immunolabeling C. elegans med antistoffer er udfordrende på grund af lav permeabilitet af enten æggeskallen eller kollagen neglebånd omkring dyret. I mellemtiden, da C. elegans proteiner er væsentligt forskellige fra deres hvirveldyr orthologs, et par kommercielle virksomheder giver C. elegans med specifikke produkter. Det er svært for en lille laboratorium til at generere C. elegans antistoffer for sig selv. Forskere i Fællesskabet bruger ofte mærket proteiner som markører til at demonstrere en protein lokalisering eller gene expression. Denne artikel bruger EKSKL-1::GFP som et eksempel til at spore en protein nukleare translokation under varme stress8. En integreret translationel ekskl-1::gfp til det animalske genom bruges til stabilt express gen med normal god landbrugspraksis fusion. Forskning har vist, at ekskl-1 udtrykkes i tarmen, kroppen væggen muskel og andre subcellulært strukturer8. Denne protokol demonstrerer, hvordan du synkroniserer orme til den fjerde larve (L4) fase, udføre en varme stress eksperiment, og adfærd DAPI farvning, ethanol fiksering, acetone fiksering og imaging under en regelmæssig 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 protokol, en grøn fluorescens protein (NGL) fusion protein og 4', 6-diamidino-2-phenylindole (DAPI) farvning bruges til at spore protein subcellulært lokalisering ændringer; især en nuklear translokation under en varme stress tilstand. Proteiner reagerer tilsvarende til ydre og indre signaler. En fælles mekanisme er at ændre sin subcellulært lokalisering. I denne artikel beskrives en protokol for at spore protein lokalisering, der ikke kræver et antistof, radioaktive mærkning eller en Konfokal mikroskop. I denne artikel bruges normal god landbrugspraksis tagge target proteinet EKSKL-1 i C. elegans, medlem af chlorid intracellulære kanal proteiner (CLICs) familie, herunder pattedyr CLIC4. En integreret translationel ekskl-1::gfp transgene linje (med en promotor og en fuld gensekvens) var lavet af transformation og γ-stråling, og udtrykker stabilt genet og normal god landbrugspraksis. Nyere forskning viste, at ved varmestress, ikke oxidativt stress, EKSKL-1::GFP ophobes i kernen. Overlappende normal god landbrugspraksis signal med både kerner struktur og DAPI signaler bekræfter EKSKL-1 subcellulært lokalisering ændringer under stress. Denne protokol udgør to forskellige fiksering metoder for DAPI farvning: ethanol fiksering og acetone fiksering. DAPI farvning protokollen præsenteret i denne artikel er hurtig og effektiv og bevarer både normal god landbrugspraksis signal og protein subcellulært lokalisering ændringer. Denne metode kræver kun et fluorescens mikroskop med Nomarski, et FITC-filteret og en DAPI filter. Det er velegnet til en lille laboratorium indstilling, bachelor studerende forskning, high school studerende forskning og bioteknologi klasseværelser.

Chlorid intracellulære kanal proteiner (CLIC) er multifunktionelle proteiner, der er særdeles bevaret på tværs af arter12. Megen forskning viser at CLICs regulere cellulære stress, autophagy, apoptose, carcinogenese, angiogenese og makrofag medfødte immunrespons i pattedyr system13,14,15,16 ,17. D...

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 protokol demonstrerer, hvordan man spore en protein nukleare translokation under varmestress ved hjælp af en grøn fluorescens protein (NGL) fusion protein som en markør og 4', 6-diamidino-2-phenylindole (DAPI) farvning. DAPI farvning protokol er hurtig og bevarer normal god landbrugspraksis og protein subcellulært lokalisering signaler.

Påvisning af Protein subcellulært lokalisering af grøn fluorescens Protein Tagging og 4', 6-Diamidino-2-phenylindole farvning 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

Grundlæggende<em&gt; Caenorhabditis elegans</em&gt; Metoder: Synkronisering og Observation

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

Forberedelse af Mgm101 Recombination Protein af MBP-baserede 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 interaktioner visualiseret ved bimolekylær Fluorescens komplementering i tobaksprotoplaster og blade

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

Grønt fluorescerende protein-baserede Expression Screening membranproteiner 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

Kvantificering af Protein Expression og Co-lokalisering Brug Multiplexed immunhistokemisk farvede og Multispektral Imaging

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

Kvantitative lokalisering af Golgi Protein af Imaging dens centrum af 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 til undersøgelse ændringer i iboende Protein sammenlægning 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 ...