Name: a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast
Uploaded: 2017-12-15T15:00:00
Description: Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

Dele af denne undersøgelse blev venligt støttet af en bevilling fra Deutsche Forschungsgemeinschaft (SFB 1027, A6).

Bemærk: En oversigt over generelle eksperimentelle arbejdsprocessen er afbildet i figur 1.
Forsigtig: RTA er stærkt giftige for mennesker. Sikkerhed lab tilladelse S2 (biosikkerhed niveau 2 tilsvarende) er nødvendig. Venligst bære handsker under hele forsøget.
1. Heterolog ekspression af hans markeret RTA i Escherichia coli
<ol>
	<li>Transformer E. coli celler med udtryk plasmidet pET24-RTA(hans) 6 el...

<ol>
	<li>Li, S., 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=Folding-competent+and+folding-defective+forms+of+ricin+A+chain+have+different+fates+after+retrotranslocation+from+the+endoplasmic+reticulum.">Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum.</a> Mol Biol Cell. 21 (15), 2543-2554 (2010).</li><li>Li, S., Spooner, R. A., Hampton, R. Y., Lord, J. M., Roberts, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosolic+entry+of+Shiga-like+toxin+a+chain+from+the+yeast+endoplasmic+reticulum+requires+catalytically+active+Hrd1p.">Cytosolic entry of Shiga-like toxin a chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p.</a> PLoS One. 7 (7), e41119 (2012).</li><li>Moreau, 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=Genome-wide+RNAi+screens+identify+genes+required+for+Ricin+and+PE+intoxications.">Genome-wide RNAi screens identify genes required for Ricin and PE intoxications.</a> Dev Cell. 21 (2), 231-244 (2011).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+toolbox+for+assessing+protein+location+and+function.">The fluorescent toolbox for assessing protein location and function.</a> Science. 312 (5771), 217-224 (2006).</li><li>Majoul, I. V., Bastiaens, P. I., Soling, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+an+external+Lys-Asp-Glu-Leu+(KDEL)+protein+from+the+plasma+membrane+to+the+endoplasmic+reticulum:+studies+with+cholera+toxin+in+Vero+cells.">Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.</a> J Cell Biol. 133 (4), 777-789 (1996).</li><li>Becker, B., Schnoder, T., Schmitt, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+Reporter+Assay+to+Identify+Cellular+Components+of+Ricin+Toxin+A+Chain+Trafficking.">Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking.</a> Toxins (Basel). 8 (12), (2016).</li><li>Li, X. P., Baricevic, M., Saidasan, H., Tumer, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+depurination+is+not+sufficient+for+ricin-mediated+cell+death+in+Saccharomyces+cerevisiae.">Ribosome depurination is not sufficient for ricin-mediated cell death in Saccharomyces cerevisiae.</a> Infect Immun. 75 (1), 417-428 (2007).</li><li>Lord, J. M., Roberts, L. M., Robertus, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ricin:+structure,+mode+of+action,+and+some+current+applications.">Ricin: structure, mode of action, and some current applications.</a> Faseb J. 8 (2), 201-208 (1994).</li><li>Becker, B., Schmitt, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adapting+yeast+as+model+to+study+ricin+toxin+a+uptake+and+trafficking.">Adapting yeast as model to study ricin toxin a uptake and trafficking.</a> Toxins (Basel). 3 (7), 834-847 (2011).</li><li>Seidman, C. E., Struhl, K., Sheen, J., Jessen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+plasmid+DNA+into+cells.">Introduction of plasmid DNA into cells.</a> Curr Protoc Mol Biol. , (2001).</li><li>Brunelle, J. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coomassie+blue+staining.">Coomassie blue staining.</a> Methods Enzymol. 541, 161-167 (2014).</li><li>Shapiro, A. L., Vinuela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.+1979.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. 1979.</a> Biotechnology. 24, 145-149 (1992).</li><li>Ito, H., Fukuda, Y., Murata, K., Kimura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+intact+yeast+cells+treated+with+alkali+cations.">Transformation of intact yeast cells treated with alkali cations.</a> J Bacteriol. 153 (1), 163-168 (1983).</li><li>Vitetta, E. S., Yen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+functional+properties+of+genetically+engineered+ricin+B+chain+lacking+galactose-binding+activity.">Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity.</a> Biochim Biophys Acta. 1049 (2), 151-157 (1990).</li><li>Wales, R., Roberts, L. M., Lord, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addition+of+an+endoplasmic+reticulum+retrieval+sequence+to+ricin+A+chain+significantly+increases+its+cytotoxicity+to+mammalian+cells.">Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells.</a> J Biol Chem. 268 (32), 23986-23990 (1993).</li><li>Breslow, D. K., 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=A+comprehensive+strategy+enabling+high-resolution+functional+analysis+of+the+yeast+genome.">A comprehensive strategy enabling high-resolution functional analysis of the yeast genome.</a> Nat Methods. 5 (8), 711-718 (2008).</li><li>Jablonowski, D., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymocin,+a+composite+chitinase+and+tRNase+killer+toxin+from+yeast.">Zymocin, a composite chitinase and tRNase killer toxin from yeast.</a> Biochem Soc Trans. 35 (Pt 6), 1533-1537 (2007).</li><li>Jablonowski, D., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+RNA+polymerase+II+is+affected+by+Kluyveromyces+lactis+zymocin.">Saccharomyces cerevisiae RNA polymerase II is affected by Kluyveromyces lactis zymocin.</a> J Biol Chem. 277 (29), 26276-26280 (2002).</li><li>Jablonowski, D., Frohloff, F., Fichtner, L., Stark, M. J., Schaffrath, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kluyveromyces+lactis+zymocin+mode+of+action+is+linked+to+RNA+polymerase+II+function+via+Elongator.">Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via Elongator.</a> Mol Microbiol. 42 (4), 1095-1105 (2001).</li></ol>

Når du udfører den ovennævnte protokol, anbefaler vi følgende forslag til at opnå et vellykket resultat af forsøget.
For heterolog protein udtryk er det vigtigt at ikke overstige IPTG koncentration af 1 mM. IPTG koncentrationer &#62; 1 mM hæmme promotor-induceret RTA udtryk og fører til lavere toksin udbytter. Desuden bør celler ikke være dyrket ved temperaturer højere end 28 ° C at forhindre inklusion krop dannelse, ineffektive foldning og toksin inaktivering. RTA udtryk ved laver...

Patienter lider af infektioner af toxin producerende bakterier udgør en alvorlig medicinsk og økonomisk byrde for hver sociale sundhedssystem, især da effektive terapeutiske behandlinger er stadig i høj grad mangler. At udvikle nye terapeutiske strategier, de komplekse forgiftning mekanismer af lægeligt relevante A / B toksiner som kolera toksin, shigatoksin eller ricin skal være fuldt forstået på det molekylære niveau baseret på romanen kraftfulde assays, der skal gennemføres.
I de seneste år, flere undersøgelser har forsøgt at analysere A / B toksin transport i gær og pattedyrceller ved hjælp af tidskrævende og omkostningstunge metoder såsom radioaktivt toksin mærkning1,2 samt siRNA-baseret screening nærmer sig3. I nogle tilfælde er toksin menneskehandel blevet visualiseret i vivo af Fluorescens mikroskopi efter kemiske og/eller genetisk kobling af individuelle toksin underenheder med fluorophores, quantum dots eller fluorescerende proteiner4,5. Desværre, sådanne ændringer ofte føre til inaktive og/eller ændrede naturlige egenskaber af toksiner. En anden elegant måde indirekte besvare en lang række videnskabelige spørgsmål er brugen af reporter systemer baseret på enzymer såsom lacZ, luciferase, eller fluorescerende proteiner (f.eks. normal god landbrugspraksis eller Discosoma sp. rødt fluorescerende proteiner ( dsRed)).
I dette manuskript, er en enkel protokol beskrevet som identificerer cellulære komponenter, der kræves til intracellulær transport af extracellularly anvendt RTA i S. cerevisiae. Dermed fungerer et fluorescens-reporter plasmid som indeholder en N-terminalen ER-import signal efterfulgt af normal god landbrugspraksis som et protein biosyntesen sensor, der indirekte måler RTA-medieret protein oversættelse hæmning af normal god landbrugspraksis fluorescens emission efter in vivo oversættelse6. I tilfælde at RTA endocytose og/eller intracellulære handel negativt (eller positivt) påvirkes i en særlig gær sletning mutant sammenlignet med wild-type, kan dette påvises gennem en stigning (eller fald) i normal god landbrugspraksis fluorescens emission6.
Hidtil har var alle metoder analysere RTA transport i gærceller begrænset til ER til cytosol retro-translokation proces af RTA. I sådan et kunstigt system udtrykkes RTA indeholdende en ER import signal fra en inducerbar promotor, hvilket resulterer i en selvmorderisk fænotype1,7. Selv om cellen bindende B-subunit af ricin mangler ligeledes i eksperimentel konfigurationen som beskrevet i dette manuskript, og således ikke fuldt ud repræsenterer naturlige situation af ricin holotoxin forgiftning8, toksin transport fra plasma membran gennem Golgi apparatet på skadestuen kan efterlignes nøje med denne roman assay. Interessant, viser de foreløbige resultater, der opnås i pilot-undersøgelse, at menneskehandel veje bruges af RTA afslører slående ligheder med ruten forgiftning af ricin holotoxin.
I sammendrag, kan den beskrevne metode bruges til at bestemme den specifikke rolle af valgte cellulære proteiner i RTA endocytose og handel med gær. Desuden, denne eksperimentelle setup kan nemt tilpasses andre ribosomet uvirksomme giftstoffer produceres og udskilles fra forskellige gær og bakteriearter zymocin eller shigatoksin.

<table>
	<tbody>
		<tr>
			<td>Bacterial and yeast strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E coli BL21 DE3(Gold)</td>
			<td>Aligent Technologies</td>
			<td>230130</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742</td>
			<td>Euroscarf</td>
			<td>Y10000</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742 deletion mutants</td>
			<td>Dharmacon</td>
			<td>YSC1054</td>
			<td>whole collection</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids used in this protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pET24a(+) (Novagen)</td>
			<td>Millipore</td>
			<td>69772-3</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-RTA(His6)</td>
			<td>Becker et al. (2016)3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 20T</td>
			<td>USBio</td>
			<td>Z1000.250</td>
			<td>lytic enzyme for cell wall removal</td>
		</tr>
		<tr>
			<td>LB broth medium</td>
			<td>Thermo Scientific</td>
			<td>10855021</td>
			<td>15 g agar was added for plate production</td>
		</tr>
		<tr>
			<td>YNB</td>
			<td>Thermo Scientific</td>
			<td>DF0335-15-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A4418-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast drop-out mix supplemts without leucine</td>
			<td>Sigma-Aldrich</td>
			<td>Y1376-20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>05040-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>10197777001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-raffinose</td>
			<td>Sigma-Aldrich</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Thermo Scientific</td>
			<td>11811031</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Roth</td>
			<td>3899.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE ruler prestained</td>
			<td>Fermentas</td>
			<td>26616</td>
			<td>protein ladder used for Western analysis</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for RTA purification, desalting and reader measurements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Ultrospec 2100 pro</td>
			<td>Amersham</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soniprep 150</td>
			<td>MSE</td>
			<td></td>
			<td>old model, other models available</td>
		</tr>
		<tr>
			<td>Fluoroskan Ascent</td>
			<td>Thermo Scientific</td>
			<td>5210470</td>
			<td>old model, not available anymore</td>
		</tr>
		<tr>
			<td>&#196;KTAPurifier</td>
			<td>Thermo Scientific</td>
			<td>28406266</td>
			<td>Product is discontinued and replaced</td>
		</tr>
		<tr>
			<td>HisTRAP HP column</td>
			<td>GE Healthcare</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTRAP desalting column</td>
			<td>GE Healthcare</td>
			<td>11-0003-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Midisart sterile filter</td>
			<td>Sartorius</td>
			<td>16534K</td>
			<td>0.2 &#181;m pore size</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>660 nm assay kit</td>
			<td>Thermo Scientific</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Thermo Scientific</td>
			<td>260860</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tetra-His</td>
			<td>Qiagen</td>
			<td>34670</td>
			<td>primary antibody; 1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Anti-mouse-HRP</td>
			<td>Sigma-Aldrich</td>
			<td>A9044-2ML</td>
			<td>secondary antibody, 1:10,000 dilution</td>
		</tr>
	</tbody>
</table>

a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast

Bakteriel og plante A / B toksiner udnytte de naturlige menneskehandel veje i eukaryote celler for at nå deres intracellulære målet (r) i cytosol og i sidste ende dræbe. Sådanne A / B toksiner generelt består af en enzymatisk aktive Asubunit (fx, ricin toksin en (RTA)) og en eller flere celle bindende Bsubunit(s), som er ansvarlige for toksin binding til specifikke celle overfladen receptorer. Vores nuværende viden om, hvordan A / B toksiner er i stand til at effektivt berusende celler hjalp forskere til at forstå grundlæggende cellulære mekanismer, som endocytose og intracellulær protein sortering i højere eukaryote celler. Fra et medicinsk synspunkt er det ligeledes vigtigt at identificere store toksin smuglerruter finder passende behandling løsninger til patienter eller i sidste ende udvikle terapeutiske toksin-baserede programmer for kræftbehandling.
Siden genome-wide analyser af A / B toksin handel i pattedyrceller er komplekse, tidskrævende og dyrt, flere undersøgelser på A / B toksin transport har været udført i gær model organisme Saccharomyces cerevisiae. Trods mindre komplekse, grundlæggende cellulære processer i gær og højere eukaryote celler kan er lignende og meget ofte resultaterne af gær blive overført til pattedyr situationen.
Her beskriver vi en hurtig og nem at bruge reporter analyse for at analysere den intracellulære handel med RTA i gær. En væsentlig fordel ved den nye assay er mulighed for at undersøge ikke kun RTA retro-translokation fra det endoplasmatiske reticulum (ER) i cytosol, men snarere endocytose og retrograd toksin transport fra plasma membran ind på Skadestuen. Analysen gør brug af en reporter plasmid, der giver mulighed for indirekte måling af RTA toksicitet via fluorescens emission af grøn fluorescerende proteiner (NGL) efter i vivo oversættelse. Da RTA forhindrer effektivt indledningen af proteinsyntese ved 28S rRNA depurination, tillader dette assay identifikation af vært celle proteiner involveret i intracellulære RTA transport gennem påvisning af ændringer i fluorescens emission.

Den generelle arbejdsgang af den protokol, der er beskrevet i dette håndskrift er illustreret i figur 1, groft sammenfatter enkelt trin til vellykket RTA rensning og efterfølgende normal god landbrugspraksis reporter assay eksperiment. En mere detaljeret beskrivelse af hvert enkelt trin kan findes i protokollen. Figur 2 illustrerer det forventede resultat af en vellykket RTA rensning af affinitet kromatografi (<strong class="xf...

Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

En simpel fluorescens-baserede Reporter analyse til at identificere cellulære komponenter kræves for Ricin toksin en kæde (RTA) handel med gær

I manuskriptet som, beskriver vi brugen af en gær-baserede fluorescens reporter analyse til at identificere blodlegemers involveret i menneskehandel og dræbe processer af den cytotoksiske en underenhed af plante toksin ricin (RTA).

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

Research

JoVE Journal

Biology

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi screening for at identificere Postembryonic fænotyper i C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Modificeret Gær-Two-Hybrid system til at identificere Proteiner Interaktion med Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolering af ribo Bundet Gryende polypeptider<em&gt; In vitro</em&gt; Til at identificere Translationelle Pause steder langs mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

En Eksplantation Assay for vurdering Cellulær Behavior af Cranial mesenkym

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

En kvantitativ analyse til at studere Protein: DNA interaktioner, Discover transskriptionsregulatorer af genekspression, og identificere nye anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

Karakterisering af G-protein-koblede receptorer med en fluorescens-baserede calciummobilisering Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

Filtrering Isolering af nukleinsyrer: en enkel og hurtig DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...