Name: characterization of multi-subunit protein complexes of human mxa using non-denaturing polyacrylamide gel-electrophoresis
Uploaded: 2016-10-28T15:00:00
Description: Watch this Scientific Journal Video about Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis at JoVE.com

This work was funded by a Grant from the Swiss National Science foundation (Grant nr. 31003A_143834) to JP.

MERK: Denne protokollen er basert på tidligere utgitt ikke-denaturering SIDE protokoll 12. I den studien ble oligomere tilstanden i MXA protein vurdert ved hjelp av enten Vero-celler som overuttrykker MXA eller IFN-a-stimulerte A549 celler som uttrykker endogen MXA. Protokollen er beskrevet nedenfor, kan brukes til å analysere den oligomere tilstand av hvilket som helst protein i tillegg til MXa. Imidlertid kan ytterligere optimalisering være nødvendig. 1. Utarbeidelse av cellel...

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Her beskriver vi en enkel fremgangsmåte som tillater rask bestemmelse av den oligomere tilstand av proteiner uttrykt i pattedyrceller ved ikke-denaturerende PAGE etterfulgt av Western blot-analyse. Den store fordelen med denne tilnærmingen er at den oligomere tilstanden til et gitt protein kan bestemmes fra helcellelysater uten forutgående proteinrensing. Dette kan være viktig for proteiner som oligomerisere eller utøver sin funksjon i forbindelse med hjelpefaktorer. I tillegg er de proteiner er fremdeles i sin opp...

Den kvaternære struktur av et protein spiller en avgjørende rolle i mange cellulære prosesser. Signalveier, genekspresjon og enzym aktivering / deaktivering alle er avhengige av riktig montering av protein komplekser 1-4. Denne prosessen også kjent som homo- eller hetero-oligomerisering er på grunn av irreversible kovalente eller reversibel elektrofile og hydrofobe protein-protein-interaksjoner. Oligomerisering ikke bare, sprer de forskjellige cellulære prosesser uten å øke genomstørrelse, men gir også en strategi for proteiner for å bygge stabile komplekser som er mer motstandsdyktig mot denaturering og degradering 5. Defekter i oligomerisering har innvirkning på funksjonen av proteiner og kan føre til utvikling av sykdommer. For eksempel, danner enzymet fenylalanin-hydroksylase et tetramert kompleks. Noen mutasjoner innenfor proteinkomplekset kan svekke tetramer formasjonen og føre til sykdommen fenylketonuri 6. <p class = &quot;jove_content&quot;&gt; Den menneskelige MXa proteinet er et interferon (IFN) -indusert antiviralt effektor-proteinet utøver en bred antiviral aktivitet mot en rekke RNA, så vel som DNA-virus 7. Den tilhører superfamily av -strøm lignende store GTPases og har kapasitet til å danne store oligomeric strukturer in vitro 8. Oligomeriseringsprosessen har blitt foreslått for å beskytte MXA fra rask nedbrytning 9,10. Til tross for intens innsats fra mange forskningsgrupper, den molekylære virkningsmekanismen fortsatt i stor grad unnvikende og rollen oligomerisering staten MXA for sin antiviral funksjon er under debatt 9,11,12. I denne forbindelse, Gao og kolleger foreslått en modell der MXA utøver sin antiviral aktivitet ved å samhandle med virus nucleoproteins i form av store ringlignende oligomeric strukturer 11. Men nå nylig, viste vi at MXA dimers utviser antiviral aktivitet og samhandle med nucleoproteins av influensa A virus 12. Based på krystallstrukturen til MXa, Gao og medarbeidere identifisert flere aminosyrerester i grensesnittet regionene som er kritiske for sin oligomerisering in vitro og dens antiviral funksjon 11,13. Derfor, for å belyse hvilken oligomere tilstand av MXa utviser antiviral aktivitet, søkte vi å etablere en enkel protokoll for å raskt bestemme oligmeric tilstanden MXa grensesnitt mutanter uttrykt i humane celler, så vel som endogen MXa uttrykt etter IFNα stimulering. Selv om det er mange teknikker som ofte brukes for å undersøke samspillet mellom proteiner som split-grønt fluorescerende protein (split-GFP) komplemente assay 14, overflate plasmon resonans 15 og Förster resonans energi overføring (FRET) 16, de gir ikke informasjon om den eksakte støkiometrien av en oligomer proteinkompleks. For undersøkelse av dette aspektet, teknikker såsommulti-vinkel lysspredning (MALS) 17 og analytisk ultracentrifugation 18 er svært nyttig. Vanligvis proteinene analysert ved hjelp av disse metodene er rensede proteiner. Oligomerisering prosesser kan også avhenge av andre cellulære faktorer. Dersom disse faktorene er ukjente, er analysen vanskeligere. I tillegg har noen proteiner er vanskelig å uttrykke i E. coli og å rense. Derfor er disse fremgangsmåter ikke er det optimale valg å analysere proteinet oligomerisering i det cellulære miljø. I tillegg er disse teknikkene krever kostbare instrumenter som ikke er lett tilgjengelig. Ikke-denaturerende polyakrylamidgel-elektroforese (PAGE), størrelseseksklusjons-kromatografi, eller kjemisk tverrbinding, etterfulgt av konvensjonell Natriumdodecylsulfat (SDS) -side er nyttige verktøy for karakteriseringen av dannelsen av oligomerer fra cellelysater 2,19,20. Disse metodene ikke krever spesialisert utstyr og kan lett performed i en standard laboratorium. Vi først evaluert forskjellige kjemiske tverrbindings protokoller som invariantly førte til ikke-spesifikk aggregering og utfelling av MXa. Derfor vi neste testet ikke-denaturerende HOVED protokoller. Som ikke-denaturering PAGE utelukker bruk av SDS, migrering av proteiner avhenger av deres opprinnelige kostnad. Blå-nativ PAGE benytter Coomassie brilliant blue G250 for å laste proteiner med en total negativ ladning, i likhet med SDS, men ikke denaturere proteinet 21. Dessverre, Coomassie brilliant blå bunnfall i nærvær av høye salter og divalente kationer (for eksempel Mg 2+) som ofte inngår i lyseringsbuffer. Avhengig av buffere som brukes, kan det være vanskelig å analysere prøven uten ytterligere optimalisering av trinn som kan ha en effekt på den oligomere proteinkompleks. Her presenterer vi en enkel protokoll basert på en tidligere publisert metode 22 for å bestemme oligomerisering avmenneskelig MXA protein fra cellelysatene ved hjelp av ikke-denaturering PAGE.

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		<tr height="84">
			<td height="84" style="height:84px;width:244px;">Slide-A-Lyzer MINI Dialysis Units, 10K MWCO, 0.5 mL</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">69570</td>
			<td style="width:268px;">Pre-equilibrate in dialysis buffer ( if Glycerol removal is desired) 
			Can be self-made according to Fiala et al. 2011</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:244px;">4&#8211;15% Mini-PROTEAN TGX Precast Protein Gels, 10-well,</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:131px;">456-1083</td>
			<td style="width:268px;">Pre-run in running buffer to adjust buffer system</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">cOmplete, Mini, EDTA-free</td>
			<td style="width:140px;">Roche&#160;</td>
			<td style="width:131px;">11836170001</td>
			<td>use 1 tablet per 50 ml</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:244px;">PBS, pH 7.4&#160; bottle a 500ml Gibco</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:244px;">Ponceau S solution</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:131px;">P7170</td>
			<td style="width:268px;">toxic! wear gloves and protect eyes!</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:244px;">NativeMark Unstained Protein Standard&#160; 50ul</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:131px;">P/N 57030</td>
			<td>load 5 ul/well</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">A549 cells</td>
			<td style="width:140px;">ATCC</td>
			<td style="width:131px;">ATCC CCL185</td>
			<td>Grow in growth medium (see Table 1)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">Vero cells</td>
			<td style="width:140px;">ATCC</td>
			<td style="width:131px;">ATCC CCL81</td>
			<td>Grow in growth medium (see Table 1)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:244px;">anti-Mx1 antibody</td>
			<td style="width:140px;">Novus Biologicals</td>
			<td style="width:131px;">H00004599_D01P</td>
			<td>Use at a 1:1000 dilution</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:244px;">ECL Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (from donkey)</td>
			<td style="width:140px;">GE-Healthcare</td>
			<td style="width:131px;">NA934V</td>
			<td>Use at a 1:10000 dilution</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:244px;">0.5% Trypsin-EDTA (1x)&#160;&#160;&#160;&#160;&#160;&#160;&#160; Life Technologies</td>
			<td style="width:140px;">Thermo Fisher</td>
			<td style="width:131px;">15400-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Iodoacetamide&#160;&#160; 5g</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:131px;">I-6125</td>
			<td>stock&#160; 100mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Bromphenolblue</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">DMEM +4.5g/l Gluc,+L-Glut,+Pyruvate life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">41966-029</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Pen&#160; Strep 100 x&#160;&#160;&#160;&#160; 100ml&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">15140 - 130</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Glutamax 100xStock, 100ml&#160;&#160;&#160;&#160; life technologies</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">350500-038</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Fetal Bovine Serum, Dialyzed , US Origin 500ml Gibco Lot:42G9552K</td>
			<td style="width:140px;">Thermo Fisher Scientific</td>
			<td style="width:131px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Cellulose filter paper</td>
			<td style="width:140px;">Bio-Rad</td>
			<td style="width:131px;">1703965</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">PVDV blotting&#160; membrane</td>
			<td style="width:140px;">GE-Healthcare</td>
			<td style="width:131px;">10600022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:140px;">Biosolve</td>
			<td style="width:131px;">0020092391BS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium fluoride (NaF)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">S-7920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">NP-40</td>
			<td style="width:140px;">Calbiochem</td>
			<td style="width:131px;">492015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">cOmplete, Mini, EDTA-free</td>
			<td style="width:140px;">Roche&#160;</td>
			<td style="width:131px;">11836170001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Tween 20</td>
			<td style="width:140px;">Calbiochem</td>
			<td style="width:131px;">6555204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">CHAPS 10% solution</td>
			<td style="width:140px;">Amresco</td>
			<td style="width:131px;">N907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">DL-Dithiothreitol (DTT)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">43819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Glycine</td>
			<td style="width:140px;">Biosolve</td>
			<td style="width:131px;">0007132391BS</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">sodium orthovanadate (Na3VO4)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">450243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Glycerol</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">G7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">b-Glycerophospate</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">G9422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Milk powder</td>
			<td style="width:140px;">Migros/Switzerland</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Methanol</td>
			<td style="width:140px;">Millipore</td>
			<td style="width:131px;">1.06009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium cloride (NaCl)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">71380</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:244px;">magnesium chloride (MgCl2)</td>
			<td style="width:140px;">Amresco</td>
			<td style="width:131px;">288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Sodium dodecyl sulphate (SDS)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">sodium hydroxide (NaOH)</td>
			<td style="width:140px;">Sigma Aldrich</td>
			<td style="width:131px;">S-8045</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of multi-subunit protein complexes of human mxa using non-denaturing polyacrylamide gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Bruk av ikke-denaturerende PAGE, analyserte vi den oligomere tilstand av det humane villtype MXa de dimere grensesnitt mutanter MXa (R640A) og MXa (L617D) samt den monomere grensesnitt mutant MXa (M527D) fra cellelysater 12. Celler ble lysert i en buffer inneholdende 1% Octylphenoxypolyethoxyethanol (NP-40) og jodacetamid for å sikre protein solubilisering og beskyttelse av frie tiolgrupper (se figur 1). Som beskrevet tidligere, ble salt og små metabolitter fjernet ved dialyse 19.</sup...

Watch this Scientific Journal Video about Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis at JoVE.com

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

Karakterisering av Multi-subenheten Protein Komplekser av menneskelig MXA bruk av ikke-denaturerende polyakrylamidgel-elektroforese

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Research

JoVE Journal

Biochemistry

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