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<table><tbody><tr><td>-80 &deg;C opslag</td><td></td><td></td><td>Voor celcultuur</td></tr><tr><td>0,1% gelatine in water</td><td>MilliporeSigma</td><td>ES-006-B</td><td>Voor celcultuur</td></tr><tr><td>0,2 &micro;m filtereenheden</td><td>Fisher</td><td>04-977-158</td><td>Voor celcultuur</td></tr><tr><td>0,5% trypsine-EDTA (10X)</td><td>ThermoFisher</td><td>15400-054</td><td>Voor celcultuur</td></tr><tr><td>1 M MgCl2</td><td>ThermoFisher</td><td>AM9010</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>1 M Tris pH 7,5</td><td>ThermoFisher</td><td>15567027</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>1 M Tris pH 8</td><td>ThermoFisher</td><td>AM9855G</td><td>Optioneel: voor het invoegen van FTA in een endogeen ribosomaal eiwitlocus</td></tr><tr><td>1,5 en 2 mL microcentrifugebuizen, geautoclaveerd</td><td></td><td></td><td>Voor transfectie</td></tr><tr><td>1,5, 2 mL en 5 mL proteïnelage-bindende microcentrifugebuizen</td><td>Eppendorf</td><td>022431081, 022431102, 0030108302</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>10 cm weefselkweekplaten</td><td></td><td></td><td>Voor celcultuur</td></tr><tr><td>10 M NaOH</td><td>MilliporeSigma</td><td>72068-100ML</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>10x Tris/glycine/SDS running buffer concentraat</td><td>Bio-Rad</td><td>1610772</td><td>Voor Western blot</td></tr><tr><td>37 &deg;C weefselkweekincubator x 2</td><td></td><td></td><td>Voor celcultuur. Een hiervan dient als de belangrijkste incubatie voor celcultuur en verlichting van getransfecteerde cellen. Tijdelijke ruimte is nodig in een tweede incubatie op de dag van ALIBi-activering om getransfecteerde cellen te huisvesten terwijl de transilluminator in de hoofdincubator wordt ingesteld.</td></tr><tr><td>37 &deg;C waterbad</td><td></td><td></td><td>Voor celcultuur</td></tr><tr><td>5 M NaCl</td><td>ThermoFisher</td><td>AM9010</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>50 mM biotin</td><td>ThermoFisher</td><td>B20656</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>6x Laemmli buffer</td><td>FIsher</td><td>50-196-784</td><td>Voor Western blot</td></tr><tr><td>70% ethanol</td><td></td><td></td><td>Voor desinfectie</td></tr><tr><td>Acetonitril</td><td>Fisher</td><td>A955</td><td>Optioneel: voor monstervoorbereiding voor massaspectrometrie</td></tr><tr><td>AcTEV proteasekit</td><td>ThermoFisher</td><td>12575015</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>ALIBi C-fragment plasmide</td><td>Addgene</td><td>#235615, 235636 &ndash; 235641</td><td>Midi-prep schaal vereist voor transfectie</td></tr><tr><td>ALIBi N-fragment plasmide</td><td>Addgene</td><td>#235614, 235635, 235642, 235643</td><td>Midi-prep schaal vereist voor transfectie</td></tr><tr><td>Aluminiumfolie</td><td></td><td></td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>ammoniumbicarbonaat</td><td>MilliporeSigma</td><td>5.3305</td><td>Optioneel: voor monstervoorbereiding voor massaspectrometrie</td></tr><tr><td>anti-FLAG antilichaam</td><td>MilliporeSigma</td><td>F3165</td><td>Voor Western blot</td></tr><tr><td>anti-GAPDH antilichaam</td><td>Cell Signaling</td><td>5174</td><td>Voor Western blot</td></tr><tr><td>anti-muis secundair antilichaam</td><td>GE Healthcare</td><td>NA931V</td><td>Voor Western blot</td></tr><tr><td>anti-konijn secundair antilichaam</td><td>GE Healthcare</td><td>NA934V</td><td>Voor Western blot</td></tr><tr><td>anti-Rpl12 antilichaam</td><td>Abcam</td><td>ab175219</td><td>Voor Western blot</td></tr><tr><td>anti-Rpl31 antilichaam</td><td>Abcam</td><td>ab103991</td><td>Voor Western blot</td></tr><tr><td>anti-Rpl5 antilichaam</td><td>ProteinTech</td><td>15430-1-AP</td><td>Voor Western blot</td></tr><tr><td>anti-V5 antilichaam</td><td>ThermoFisher</td><td>R960-25</td><td>Voor Western blot</td></tr><tr><td>Apyrase</td><td>NEB</td><td>M0398L</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>Beta-mercaptoethanol (55 mM)</td><td>ThermoFisher</td><td>21985-023</td><td>Voor celcultuur</td></tr><tr><td>Blauw licht transilluminator</td><td>Clare Research Chemical</td><td>DR46B (klein) of DR196 (groot)</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>Rundserumalbumine (BSA)</td><td>Fisher</td><td>BP1600-100</td><td>Voor Western blot</td></tr><tr><td>C18 tips (OMIX C18 tips)</td><td>Agilent</td><td>A57003100</td><td>Optioneel: voor monstervoorbereiding voor massaspectrometrie</td></tr><tr><td>Celvriescontainer</td><td></td><td></td><td>Voor celcultuur</td></tr><tr><td>Centrifuge geschikt voor 15 mL en/of 50 mL conische buizen</td><td></td><td></td><td>Voor celcultuur</td></tr><tr><td>Centrifuge voor 1,5-2 mL microcentrifugebuizen</td><td></td><td></td><td>Voor lysis &amp; affiniteitszuivering. Moet in staat zijn tot koeling tot 4 C en snelheden tot 17.000 g. Idealiter bevindt het zich in een kamer die tijdelijk kan worden verduisterd tijdens het overbrengen van buizen in en uit.</td></tr><tr><td>Clarity ECL kit</td><td>Bio-Rad</td><td>170-5061</td><td>Voor Western blot</td></tr><tr><td>Criterion geltank</td><td>Bio-Rad</td><td>1656001</td><td>Voor Western blot</td></tr><tr><td>Criterion TGX voorgevormde gels, 4-20%</td><td>Bio-Rad</td><td>5671095</td><td>Voor Western blot. Aanvullende welformaten en lay-outs beschikbaar</td></tr><tr><td>Cycloheximide</td><td>MilliporeSigma</td><td>C7698-5G</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>Deoxycholaat</td><td>MilliporeSigma</td><td>S1827</td><td>Voor lysis &amp; affiniteitszuivering</td></tr><tr><td>Dithiothreitol</td><td>ThermoFisher</td><td>PIA39255</td><td>Optioneel: voor monstervoorbereiding voor massaspectrometrie</td></tr><tr><td>DPBS, zonder calcium, zonder magnesium</td><td>ThermoFisher</td><td>14190-250</td><td>Voor celcultuur</td></tr><tr><td>ES-gekwalificeerd FBS</td><td>MilliporeSigma</td><td>ES-009-B</td><td>Voor celcultuur</td></tr><tr><td>Ethanol</td><td>Gold Shield</td><td>412828

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

Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation

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

Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of ...

Our research investigates how ribosome composition and mRNA translation vary across subcellular domains to regulate local protein synthesis. Current methods lack spatial and temporal precision. ALIBi addresses this through optogenetically controlled proximity labeling of ribosomes.To begin, obtain L31FTA mouse embryonic stem cells and transfect them with ALIBi, GFP control, or full length BirA plasmids. After 24 to 48 hours of transfection, pre-chill the centrifuge to four degrees Celsius. With the lights off, move the transfected plates to a temporary 37-degree Celsius incubator.To prevent temperature fluctuations, once the transilluminator is placed in the main incubator, using 70%ethanol, wipe down the blue light transilluminator. After connecting the transilluminator to power, place it in the main incubator. Allow the incubator conditions to reequilibrate, while continuing the setup.Warm an aliquot of media to 37 degrees Celsius for dosing with biotin and cyclohexamide, or CHX. If preparing control samples without supplemental biotin, warm a separate aliquot for CHX-only dosing. Chill an aliquot of DPBS intended for CHX dosing.Prepare the RNase removal buffer, salt wash, lysis buffer, and wash buffers. Using a pipette, mix buffers containing dense or viscous components to ensure even mixing. Chill the prepared buffers on ice before use.Re-suspend the stock bottle of streptavidin beads by gentle pipetting or rocking. Transfer 450 microliters of beads into a protein low binding tube for each 10 centimeter plate of cells. Place the re-suspended beads on the magnetic rack for one minute, or until the beads have fully migrated.Aspirate and discard the bead storage buffer without touching the bead pellet. Wash the beads two times with 900 microliters of RNAs removal buffer. Wash the beads once with 900 microliters of salt wash.Wash the beads two times with 900 microliters of wash I buffer. Store the beads in the wash I buffer at four degrees Celsius while performing activation and cell lysis. Add biotin and CHX to the warmed aliquot of media and keep at 37 degrees Celsius until use.Add CHX to the cold DPBS aliquot and keep it chilled until use. Aspirate the media from the transfected plates. Immediately add 10 milliliters of warm media containing CHX with or without Biotin to each plate.Place negative control plates in a dark incubator. Place the remaining plates directly on top of the transilluminator. Turn on the transilluminator and incubate the plates for 15 minutes.Wash each plate two times with the 10 milliliters of cold DPBS containing CHX. Place the plates on ice between and after washes. After aspirating the second wash, tilt the plate and aspirate any residual DPBS.Keeping the plate flat on ice, add two milliliters of ice-cold lysis buffer and incubate the plate on ice for five minutes. Tap the plate two to three times during incubation to dislodge cells. Using a micropipette, pipette the lysate up and down across the plate surface to dislodge any remaining cells.Transfer the lysate to a chilled labeled protein low binding tube. In difficult to lyse organelles, sonicate all samples for 45 seconds at four degrees Celsius. And if required, transfer the lysate into a larger tube before sonication.Transfer the sonicated lysate into a two-milliliter tube. Incubate the lysate on ice for 15 minutes. Centrifuge the lysate at 17, 000 G for three minutes at four degrees Celsius.Transfer the supernatant into a chilled protein low binding tube without disturbing the pellet. Using a magnetic rack, pellet the washed strepped avident beads. Aspirate the wash one buffer.Transfer 1, 200 microliters of clarified lysate to the tube containing the washed beads. Pipette up and down gently to re-suspend the beads. Store the remaining clarified lysate at minus 80 degrees Celsius.Incubate the lysate with the beads at four degrees Celsius in the dark on a tube rotator for one hour. During the incubation, prepare the Tobacco Etch Virus, or TEV elution buffer. Pellet the beads on a magnetic rack.Transfer the supernatant to a chilled protein low binding tube. Store the supernatant at minus 80 degrees Celsius. Wash the beads two times with 900 microliters of wash I buffer and once with 900 microliters of wash II buffer.Add 450 microliters of TEV elution buffer to the beads. Incubate the beads in TEV elution buffer for one hour at room temperature on a tube rotator. Pellet the beads on the magnetic rack.Transfer the elute to a protein low binding tube. Store the eluate at minus 80 degrees Celsius if proteomics analysis or Western blot is intended. Cells containing all system components showed a L31-FTA band, which was stronger with full length burray and faint or absent when components were omitted.Endogenous biotinilated carboxylase proteins were detected in all lysates at 70 to 80 kilodaltons and 120 to 130 kilodaltons. ANTV5 probing confirmed expression of the split bore fragments. Biotinillated L31FTA decreased in the flow through relative to the lysate.No biotinillated L31-FTA was present in the eluate following TEV-mediated elution. Anti-flag probing showed a cleaved L31-FTA band at a lower molecular weight than intact L31-FTA ribosomal protein. S12 probing demonstrated the presence of the small ribosomal subunit in the eluate.Anti-V5 probing showed that proteins that do not bind the ribosome were absent from the eluate. When 2.5 fold more lysate was used with the same 450 microliters of streptavidin coated beads, a substantial amount of biotinilated L31-FTA remained in the flow through. Endogenous biotinilated proteins were partially depleted in the flow through.Endogenous biotinillated proteins were absent from the eluet, but were recovered after heating post-dilution beads in lambly buffer. Anti-flag probing detected cleaved L31-FTA at a lower molecular weight than intact L31-FTA. Anti-V5 probing showed that the split burray fragments were absent from the eluate.ALIBi enables the identification of ribosome-bound proteins and mRNAs in specific subcellular regions, revealing differences in translation regulation. The key challenge is maintaining specific proximity labeling through careful control of targeting construct expression and illumination conditions. Following this protocol, affinity purified ribosomes and associated mRNAs can be analyzed using quantitative proteomic and transcriptomic approaches.

isolation-subcellular-ribosome-subpopulations-based-on-recombinant

Research

JoVE Journal

Biology

Biologie

Een belangrijke belemmering voor de biochemische analyses van de ribosomen met ontluikende peptidyl-tRNA's is de aanwezigheid van andere ribosomen zijn in dezelfde monsters, ribosomen niet betrokken bij de vertaling van de specifieke mRNA-sequentie worden geanalyseerd. We ontwikkelden een eenvoudige methode om uitsluitend te zuiveren,, de ribosomen met de ontluikende peptidyl-tRNA van belang.

Isolatie van vertalen Ribosomen bevattende peptidyl-tRNA&#39;s voor functionele en structurele analyses

Een techniek om translationeel pauze sites op mRNA te identificeren wordt beschreven. Deze procedure is gebaseerd op isolatie van beginnende Polypeptiden ophopen op ribosomen tijdens in vitro translatie van een doel-mRNA, gevolgd door de grootte analyse van de ontluikende ketens met een denaturerende gelelektroforese.

Isolatie van Ribosoom Bound Nascent Polypeptiden<em&gt; In vitro</em&gt; Om Translationeel Pauze plaatsen langs mRNA identificeren

We beschrijven hier een techniek die nu standaard wordt gebruikt om stabiel gebonden ribosoom ontluikende keten complexen (RNC) te isoleren. Deze techniek maakt gebruik van de ontdekking dat een 17 aminozuur lange SecM "arrestatie sequentie" kan vertaling rek halt toe te roepen in een prokaryotische...

Met behulp van SecM Arrestatie sequentie als een Tool om Ribosoom Bound Polypeptiden Isoleer

Technieken om de mechanismen die ten grondslag liggen aan de afscheiding van HIV-1 Nef in exosomes ontleden worden beschreven. Specifieke korte peptiden afgeleid van Nef en eiwit transfectie werden uitgebuit om de structuur, functie, en bindende partners van Nef's Secretion Wijziging Region bepalen. Deze procedures hebben algemene belang in veel mechanistische studies.

Peptide-gebaseerde identificatie van functionele Motieven en hun bindingspartners

Dit protocol illustreert wezenlijke modificaties van polyribosome fractionering om de translatome in vivo CZS monsters te bestuderen. Het maakt het mogelijk globale beoordeling van vertaling en transcriptie regulatie door de isolatie en vergelijking van de totale RNA gebonden RNA fracties ribosoom.

In vivo Ondervraging van het centrale zenuwstelsel Translatome door Polyribosome Fractionation

We beschrijven hier een methode voor de identificatie van klein molecuul-bindende eiwitten met behulp van fotoaffiniteitslabeling. Het voordeel van deze techniek is dat binding en covalente labeling van de doeleiwitten plaatsvindt binnen de levende cellulaire omgeving, waardoor het risico van verstoring van de native eiwitstructuur en bindingscondities bij cellyse wordt geëlimineerd.

Identificatie van kleine synthetische molecules bindende eiwitten in een Inheemse cellulaire omgeving door live-cell fotoaffiniteitslabeling

We presenteren een methode voor de zuivering, detectie en identificatie van diGly peptiden die afkomstig zijn van ubiquitinated eiwitten uit complexe biologische monsters. De gepresenteerde methode is reproduceerbaar, robuust en presteert beter dan gepubliceerde methoden met betrekking tot het diepteniveau van de ubiquitinomenanalyse.

Detectie van eiwitubiquitinatiesites door peptideverrijking en massaspectrometrie

Hier beschrijven we de immunopreciatie van riosomen en bijbehorend RNA uit geselecteerde populaties volwassen mannelijke muizenkiemcellen met behulp van het RiboTag-systeem. Strategische veredeling en zorgvuldige immunoneerslag resulteren in schone, reproduceerbare resultaten die informeren over het vertalen van de kiemcel en inzicht geven in de mechanismen van gemuteerde fenotypes.

RiboTag immunoprecipitatie in de kiemcellen van de mannelijke muis

Single molecule fluorescentie energieoverdracht is een methode die de tRNA-dynamiek volgt tijdens ribosomale eiwitsynthese. Door individuele ribosomen te volgen, worden inhomogene populaties geïdentificeerd, die licht werpen op mechanismen. Deze methode kan worden gebruikt om biologische conformatieveranderingen in het algemeen te volgen om dynamisch-functierelaties in vele andere complexe biosystemen te onthullen. Single molecule-methoden kunnen niet-snelheidsbeperkende stappen en laagbevolkte...

Single Molecule Fluorescence Energy Transfer Study van ribosoom eiwitsynthese

Co-translationele interacties spelen een cruciale rol bij ontluikende ketenaanpassingen, targeting-, vouw- en assemblagepaden. Hier beschrijven we Selective Ribosome Profiling, een methode voor in vivo, directe analyse van deze interacties in het model eukaryote Saccharomyces cerevisiae.

Wereldwijde identificatie van co-translationele interactienetwerken door selectieve ribosoomprofilering

We presenteren een tweestappenprotocol voor hoogwaardige mitochondrische isolatie dat compatibel is met eiwitontdekking en kwantificering op proteoomschaal. Ons protocol vereist geen genetische manipulatie en is dus geschikt voor het bestuderen van mitochondriën uit primaire cellen en weefsels.

Tweestaps tagvrije isolatie van mitochondriën voor verbeterde eiwitontdekking en kwantificering

Stanford School of Medicine.  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

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