Name: Verrijken van subcellulaire eiwitten in leptospira met behulp van een op Triton X-114 gebaseerde fractioneringsbenadering (Video) | JoVE Journal
Uploaded: 2025-08-08T12:30:00.000+00:00
Duration: 4 min 25 s
Description: Leer subcellulaire eiwitfractie in Leptospira met Triton X-114 en massaspectrometrie. Deze methode identificeert virulentiefactoren en verbetert het begrip van pathogene bacteriële proteomics.

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<li>Madathiparambil, M. G., Cattavarayane, S., Perumana, S. R., Manickam, G. D., Sehgal, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+46+kDa+gelatinase+on+the+outer+membrane+of+Leptospira%5BTitle%5D)+AND+%22Curr+Microbiol%22%5BJournal%5D)">Presence of 46 kDa gelatinase on the outer membrane of Leptospira.</a> Curr Microbiol. 62 (5), 1478-1482 (2011).</li>
</ol>

<table><tbody><tr><td>Azijnzuur</td><td>&nbsp; Sigma</td><td>&nbsp;695092</td><td></td></tr><tr><td>Aceton</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;32201</td><td></td></tr><tr><td>Acrylamid</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;A-3553</td><td></td></tr><tr><td>Ammoniumpersulfaat</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;A-3678</td><td></td></tr><tr><td>Balans</td><td>&nbsp;SHIMADUZ</td><td>&nbsp;Z741079</td><td></td></tr><tr><td>Bis Acrylamide</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;M7279</td><td></td></tr><tr><td>Rundserumalbumine fractie V (BSA)</td><td>&nbsp; Sigma</td><td>&nbsp;A-8022</td><td></td></tr><tr><td>Broomfenolblauw</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;B-8026</td><td></td></tr><tr><td>Centrifugebuis (50 mL)</td><td>Tarsons</td><td>500020</td><td></td></tr><tr><td>Conische fles met dop (150 mL)</td><td>Tarsons</td><td>431030</td><td></td></tr><tr><td>Koelcentrifuge</td><td>&nbsp;Eppendorf</td><td>&nbsp;5355316200</td><td></td></tr><tr><td>Coomassie briljantblauw</td><td>&nbsp;&nbsp;Sigma</td><td>1.12553</td><td></td></tr><tr><td>Donkerveldmicroscoop</td><td>&nbsp;ZEISS</td><td>&nbsp;GmbH37081</td><td></td></tr><tr><td>Delicate Task Wipes</td><td>Kimtech Science</td><td>34155</td><td></td></tr><tr><td>Droogbad</td><td>&nbsp;Eppendorf</td><td>&nbsp;535531620</td><td></td></tr><tr><td>Ellinghausen&ndash;McCullough&ndash;Johnson&ndash;Harris (EMJH) medium, Difco</td><td>&nbsp;Difco</td><td>&nbsp;279410</td><td></td></tr><tr><td>Gellades: Gellades moeten minimaal 2 cm breder zijn in afmetingen en een glad en vlak oppervlak hebben met deksel. </td><td>&nbsp;Hoefer</td><td>&nbsp;Niet van toepassing</td><td></td></tr><tr><td>Glasbeker (150 mL)</td><td>Tarsons</td><td>421030</td><td></td></tr><tr><td>Glycine G8790</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;G-8898</td><td></td></tr><tr><td>Immobilon Blotting Filter Paper</td><td>Millipore</td><td>IBFP0813C</td><td></td></tr><tr><td>Immunoblot semi-droge opstelling</td><td>&nbsp;ATTA</td><td>&nbsp;AE-6675</td><td></td></tr><tr><td>Incubator - Voor het incuberen van de gellades gedurende 18 uur bij 37&deg;C.</td><td>&nbsp;EYELA</td><td>&nbsp;SLI-700</td><td></td></tr><tr><td>Laminale luchtstroom</td><td>&nbsp;Niet van toepassing</td><td>&nbsp;Niet van toepassing</td><td></td></tr><tr><td>Luminol</td><td>&nbsp; Sigma</td><td>&nbsp;09253</td><td></td></tr><tr><td>Magnesiumchloride M8266</td><td>&nbsp; Sigma</td><td>&nbsp;M8266</td><td></td></tr><tr><td>Magneetschep</td><td>&nbsp;TARSONS</td><td>&nbsp;6040</td><td></td></tr><tr><td>Methanol</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;17995-7</td><td></td></tr><tr><td>Micropipettipjes (10 &micro;L&ndash;10 mL)</td><td>Axygen</td><td>T-10, T-200-C,T-1000-C, T-10ML-C</td><td></td></tr><tr><td>Micropipetten</td><td>&nbsp;Eppendorf</td><td>&nbsp;EP2231300010</td><td></td></tr><tr><td>Microtubes (1,5 mL Safe-Lock)</td><td>Eppendorf</td><td>EP022363212</td><td></td></tr><tr><td>Mini spin</td><td>&nbsp;Genaxy</td><td>&nbsp;GEN-MINI-6K</td><td></td></tr><tr><td>Multimode reader</td><td>BMG LABTECH</td><td>&nbsp;S/N 413-3877</td><td></td></tr><tr><td>Vrij van vet droge melk, rund</td><td>Sigma-Aldrich</td><td>M7409</td><td></td></tr><tr><td>P-Coumaric acid</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;C9008</td><td></td></tr><tr><td>Fosfaat gebufferde zoutoplossing</td><td>&nbsp;&nbsp;Sigma</td><td>P4417&nbsp;</td><td></td></tr><tr><td>Koelkast</td><td>&nbsp;LG</td><td>&nbsp;GC-B217BLJ2</td><td></td></tr><tr><td>Rocker - Roterende schudler met lage snelheid voor het kleuren van de gel.</td><td>&nbsp; TARSONS</td><td>&nbsp;4080</td><td></td></tr><tr><td>SDS-PAGE SETUP- Electroforese Apparatuur</td><td>&nbsp;Hoefer, Inc.</td><td>&nbsp;SE300-10A-1.0</td><td>&nbsp;Hoefer model &lsquo;miniVE&rsquo; die 10 cm x 8 cm grote gel van dikte 1 mm kan gieten. </td></tr><tr><td>Natriumchloride</td><td>&nbsp;&nbsp;&nbsp;Sigma</td><td>&nbsp;5886</td><td></td></tr><tr><td>Natriumdodecylsulfaat (SDS)</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;L-4396</td><td></td></tr><tr><td>TEMED</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;T-9281</td><td></td></tr><tr><td>Triton X-114</td><td>&nbsp;&nbsp;Sigma</td><td>648468</td><td></td></tr><tr><td>Trizma Base (TRIS) T6066</td><td>&nbsp;&nbsp;Sigma</td><td>&nbsp;T6066</td><td></td></tr><tr><td>Vortex</td><td>&nbsp;TARSONS</td><td>&nbsp;3020</td><td></td></tr></tbody></table>

verrijken van subcellulaire eiwitten in leptospira met behulp van een op triton x-114 gebaseerde fractioneringsbenadering

Enriching Subcellular Proteins in Leptospira Using a Triton X-114-Based Fractionation Approach

Verrijken van subcellulaire eiwitten in leptospira met behulp van een op Triton X-114 gebaseerde fractioneringsbenadering

Prokaryotes lack precise subcellular organelles but exhibit distinct regions such as cytoplasm, inner membrane, periplasm, and outer membrane, where most ...

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Biochemistry

Biochemie

Affiniteitszuivering van gemerkte eiwitten in combinatie met massaspectrometrie (APM) is een krachtige methode voor systematische mapping van eiwit interactie netwerken en het onderzoeken van de mechanistische basis van biologische processen. Hier beschrijven we een geoptimaliseerde sequentiële peptide affiniteit (SPA) APM procedure ontwikkeld voor de...

Identificatie van eiwit complexen in<em&gt; Escherichia coli</em&gt; Met Sequential Peptide Affinity Purification in combinatie met tandem massaspectrometrie

Wij presenteren een variatie van de QUICK (kwantitatieve Immunoprecipitatie combinatie met Knockdown) benadering die eerder werd onderscheid te maken tussen echte en valse eiwit-eiwit interacties. Onze aanpak is gebaseerd...

Een protocol voor de identificatie van eiwit-eiwit interacties op basis van<sup&gt; 15</sup&gt; N Metabole Labeling, Immunoprecipitatie, kwantitatieve Massaspectrometrie en Affiniteit Modulatie

Source: Armbruster, K. M., Meredith, T. C. Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry. J. Vis. Exp. (2018)This video demonstrates the enrichment of bacterial lipoproteins using a non-ionic detergent that undergoes phase separation upon warming. Through sequential temperature shifts and centrifugation steps, lipoproteins are selectively partitioned into the detergent phase, while non-lipoprotein contaminants...

Enrichment of Bacterial Lipoproteins Using Non-Ionic Detergent Phase Separation

Biofysische en biochemische studies van interacties tussen membraan-ingebed eiwitdomeinen geconfronteerd met vele technische uitdagingen, waarvan de eerste is het verkrijgen van passende studiemateriaal. Dit artikel beschrijft een protocol voor het produceren en zuiveren van disulfide-gestabiliseerde transmembraan peptide complexen die geschikt zijn voor structurele analyse door oplossing kernspinresonantie (NMR) en andere analytische toepassingen.

Productie van Disulfide gestabiliseerde Transmembrane peptidecomplexen voor structurele studies

Een biochemische benadering wordt beschreven in vivo eiwit-eiwit interacties (PPI) van membraaneiwitten identificeren. De methode combineert eiwit verknoping, affiniteitszuivering en massaspectrometrie, en is aanpasbaar aan vrijwel elk type cel of organisme. Met deze benadering ook de identificatie van voorbijgaande PPI mogelijk.

Membraan-STEKEL: een biochemische Tool om eiwit-eiwit interacties van membraaneiwitten Identificeer In Vivo

De verrijking van bacteriële lipoproteins met behulp van een fase van de niet-ionogene oppervlakteactieve stof partitioneren methode wordt beschreven voor direct gebruik in TLR testen of andere toepassingen. Verdere stappen zijn gedetailleerd voor te bereiden al lipopeptides van N-terminale structurele karakterisering door massaspectrometrie.

Verrijking van Lipoproteins van de bacteriële en de bereiding van N-terminal Lipopeptides voor structurele bepaling door massaspectrometrie

Een protocol voor de winning van een chaperone periplasmische overgangsmetalen in het kader van haar eigen bindende partners, en biofysische karakterisering van de inhoud van het substraat door Röntgen fluorescentie en radiometal opname wordt gepresenteerd.

Essentieel metalen opname bij gram-negatieve bacteriën: Röntgen fluorescentie, radio-isotopen, en cel fractionering

Dit protocol beschrijft technieken gebruikt om te bepalen van ion kanaal structuren door cryo-elektronenmicroscopie, met inbegrip van een systeem van de baculovirus gebruikt om genen in zoogdiercellen met minimale inspanning en toxiciteit, eiwit winning, zuivering, efficiënt en kwaliteit controleren, Raster monstervoorbereiding en screening, evenals gegevensverzameling en verwerking.

Meningsuiting en zuivering van de TRPC3 mens lipide-gevoelige catie kanaal voor structurele bepaling door Single-deeltje Cryo-elektronenmicroscopie

Hier bieden we een gedetailleerd protocol om te isoleren van kleine extracellulaire blaasjes (EVs) hele weefsel, met inbegrip van hersenen en tumor exemplaren. Deze methode biedt een reproduceerbare techniek om EVs extract van solide weefsel voor verder stroomafwaarts analyses.

Winning van extracellulaire blaasjes uit hele weefsel

Dit protocol beschrijft grootte-uitsluitingschromatografie, een gemakkelijke en reproduceerbare techniek voor het verrijken van Mycobacterium tuberculosis extracellulaire blaasjes uit kweeksupernatanten.

Mycobacterium tuberculose Extracellulaire vesikelverrijking door grootte-uitsluitingschromatografie

AviTag-specifieke locatie-beperkte verlichtingsversterkte biotinylatie (ALIBi) is een nieuwe optogenetisch geactiveerde methode ontwikkeld voor het labelen en affiniteitszuiveren van subcellulaire populaties van ribosomen voor downstream sequencing en proteomische analyse.

Isolatie van subcellulaire ribosoomsubpopulaties op basis van recombinante peptide-tag-specifieke locatie-beperkte verlichtingsversterkte biotinylering

Leer subcellulaire eiwitfractie in Leptospira met Triton X-114 en massaspectrometrie. Deze methode identificeert virulentiefactoren en verbetert het begrip van pathogene bacteriële proteomics.

Verrijken van subcellulaire eiwitten in leptospira met behulp van een op Triton X-114 gebaseerde fractioneringsbenadering

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