Name: Monitoring van conformationele dynamica van enkelvoudige ongemodificeerde eiwitten met behulp van plasmonische nanopincetten (Video) | JoVE Journal
Uploaded: 2025-03-21T15:00:00.000+00:00
Duration: 9 min 33 s
Description: Leer plasmonische nanotangentechnieken voor real-time monitoring van ongemodificeerde eiwitten op enkelvoudig molecuulniveau. Deze methode onthult conformatiedynamica relevant voor ziekten zoals Alzheimer en Parkinson.

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</ol>

<table><tbody><tr><td>100x Objective (NA = 1.25)</td><td>Olympus</td><td>PLN100XO</td><td>Reinig uitsluitend met lensreinigingsdoek in één beweging</td></tr><tr><td>12-1 rotary bidirectional microfluidic valve</td><td>Elveflow</td><td></td><td></td></tr><tr><td>3/2 way solenoid valve</td><td>Elveflow</td><td></td><td></td></tr><tr><td>4x Objective (NA = 0.1)</td><td>Olympus</td><td>PLN4XP</td><td></td></tr><tr><td>Airgun</td><td>RS Components</td><td>666-6772</td><td></td></tr><tr><td>Avalanche photodiode (APD)</td><td>Thorlabs</td><td>APD120A/M</td><td>Niet oververzadigd, 50 MHz bandbreedte, statisch gevoelig</td></tr><tr><td>Butterfly laser diode</td><td>Thorlabs</td><td>FPL852S</td><td></td></tr><tr><td>Charge-coupled device (camera)</td><td>Hikrobot</td><td>MV-CE200-10UC</td><td></td></tr><tr><td>Crystallising dish</td><td>VWR</td><td>216-0065</td><td></td></tr><tr><td>Data acquisition card&nbsp;</td><td>National instruments</td><td>USB-6361</td><td>2 MHz bemonsteringssnelheid</td></tr><tr><td>Double-sided tape</td><td>Adhesive Research Inc</td><td>ARcare92712</td><td>Snij de geschikte vorm voor de flow cell met een lasersnijder</td></tr><tr><td>Ethanol</td><td></td><td></td><td>Alleen in een afzuigkap, gebruik handschoenen</td></tr><tr><td>Fibreport collimator</td><td>Thorlabs</td><td>PAF2-A7B</td><td></td></tr><tr><td>Glass microscope coverslips (thickness 0.17 mm)</td><td></td><td></td><td></td></tr><tr><td>Half wave plate</td><td>Thorlabs</td><td>AHWP10M-980</td><td></td></tr><tr><td>Ideal-tek 120 mm, stainless steel, straight tweezers</td><td>RS Components</td><td>282-7472</td><td>Voor het vasthouden van monsters en het afpellen van dubbelzijdig plakband</td></tr><tr><td>Isopropanol (IPA)</td><td></td><td></td><td>Alleen in een afzuigkap, gebruik handschoenen</td></tr><tr><td>Lens cleaning tissue</td><td>Thorlabs</td><td>MC-5</td><td></td></tr><tr><td>Metrosil rapid duplicating silicone parts A and B</td><td>Metrodent</td><td>MSILR/1&nbsp;</td><td></td></tr><tr><td>Microscope slides (75 mm x 26 mm)</td><td></td><td></td><td></td></tr><tr><td>Modular Optical Tweezers System</td><td>Thorlabs</td><td>OTKB/M-CUSTOM</td><td></td></tr><tr><td>Objective oil</td><td>Olympus</td><td></td><td>Gebruik handschoenen, bewaar tussen 2oC-8oC</td></tr><tr><td>Photodiode laser mount</td><td>Thorlabs</td><td>CLD1015</td><td></td></tr><tr><td>Piezoelectric controller</td><td>Thorlabs</td><td>MDT693B</td><td></td></tr><tr><td>Piezoelectric stage</td><td>Thorlabs</td><td>Nanomax 300</td><td></td></tr><tr><td>Planoconcave lens (f = -50 mm)</td><td>Thorlabs</td><td>LC1715-B</td><td></td></tr><tr><td>Planoconvex lens (f = 150 mm)</td><td>Thorlabs</td><td>LA1433-B</td><td></td></tr><tr><td>Planoconvex lens (f = 60 mm)</td><td>Thorlabs</td><td>LA1134-B</td><td></td></tr><tr><td>Poly(ethylene glycol) methyl ether thiol (PEG-thiol) 800 MW</td><td>Sigma Aldrich</td><td>729108</td><td>Bewaar tussen 2oC-8oC</td></tr><tr><td>PTFE tubing (ID: 0.18 mm)</td><td>Vici Jour</td><td>JR-T-6805-M25</td><td></td></tr><tr><td>PTFE tubing (ID: 0.8 mm)</td><td>Cole-Parmer</td><td>WZ-21942-72</td><td></td></tr><tr><td>SEM-FIB</td><td>Zeiss</td><td>Crossbeam 550</td><td>Gallium ionenbron</td></tr><tr><td>Shortpass dichroic mirror 805 nm</td><td>Thorlabs</td><td>DMSP805</td><td></td></tr><tr><td>Silver mirror</td><td>Thorlabs</td><td>PF-10-03-P01</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>70-4511</td><td></td></tr><tr><td>Weller Erem 120 mm stainless steel, flat, rounded tweezers</td><td>RS Components</td><td>176-1150</td><td>Voor het vasthouden van een glazen deksel en het afpellen van lijm van de flow cell</td></tr></tbody></table>

monitoring van conformationele dynamica van enkelvoudige ongemodificeerde eiwitten met behulp van plasmonische nanopincetten

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Monitoring van conformationele dynamica van enkelvoudige ongemodificeerde eiwitten met behulp van plasmonische nanopincetten

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...

monitoring-conformational-dynamics-single-unmodified-proteins-using

Research

JoVE Journal

Engineering

Techniek

Plasmonische pincet en fotonische kristal nanostructuren worden getoond om nuttige verbeteringen te produceren in de efficiëntie en de oriëntatie controle van optisch trapping micro-en nano-deeltjes.

Gebruik van plasmonische en fotonisch kristal nanostructuren voor Enhanced micro-en nanodeeltjes Manipulatie

In dit artikel beschrijven we het gebruik van magnetische pincet om het effect van geweld op enzymatische proteolyse studeren aan de enkel molecuul niveau in een zeer parallelizable manier.

Multiplexed Single-molecule force Proteolyse metingen met behulp van magnetisch pincet

Hier beschrijven we de instrumenten en werkwijzen voor het detecteren enkele fluorescentie-gemerkt eiwitmoleculen interactie met een enkel DNA-molecuul opgehangen tussen twee optisch gevangen microsferen.

De combinatie van Single-molecule Manipulatie en Imaging voor de Studie van eiwit-DNA interacties

Optische pincetten zijn gebruikt om RNA-vouwing te bestuderen door individuele moleculen uit te rekken vanaf hun 5’ en 3’ uiteinden. Hier worden gebruikelijke procedures beschreven voor het synthetiseren van RNA-moleculen voor tweezing, kalibratie van het instrument en methoden om enkele moleculen te manipuleren.

Nanomanipulatie van Single RNA-moleculen door een optisch pincet

Een microchip fabricageproces dat plasmonische pincet opgenomen wordt hier gepresenteerd. De microchip maakt het afbeelden van een gevangen deeltje trapping maximale krachten meten.

Plasmonische Trapping en het vrijkomen van nanodeeltjes in een Monitoring Milieu

We beschrijven de gedetailleerde procedures en de strategieën voor het meten van de mechanische eigenschappen en mechanische ontvouwen trajecten van één eiwitmolecules met behulp van een atomic force microscope. We tonen ook representatieve resultaten als een referentie voor selectie en motivering van goed één eiwit molecuul opnames.

Kracht spectroscopie van één eiwitmolecules met behulp van een Atomic Force Microscope

We beschrijven de toepassing van infrarood nano spectroscopie en hoge-resolutie atomaire kracht microscopie om het proces van eiwit zelf montage te visualiseren in oligomere aggregaten en amyloïde fibrils, die nauw verbonden is met het ontstaan en de ontwikkeling van een breed scala aan humane neurodegeneratieve aandoeningen.

Karakteriseren van individuele eiwit aggregaten door infrarood nano spectroscopie en Atoom kracht microscopie

Dit protocol presenteert een complete experimentele workflow voor het bestuderen van RNA-eiwitinteracties met behulp van optische pincetten. Verschillende mogelijke experimentele opstellingen worden geschetst, waaronder de combinatie van een optisch pincet met confocale microscopie.

Optisch pincet om RNA-eiwitinteracties in vertaalregelgeving te bestuderen

Tijd-opgeloste single-molecule eiwit-geïnduceerde fluorescentieverbetering is een nuttige fluorescentiespectroscopische nabijheidssensor die gevoelig is voor lokale structurele veranderingen in eiwitten. Hier laten we zien dat het kan worden gebruikt om stabiele lokale conformaties in α-Synuclein te ontdekken, dat ook wel bekend staat als bolvormig ongestructureerd en onstabiel wanneer het wordt gemeten met behulp van de FRET-liniaal met een groter bereik.

Gebruik van time-resolved eiwit-geïnduceerde fluorescentieverbetering om stabiele lokale conformaties één α-synucleïnemonomeer tegelijk te identificeren

Het huidige protocol beschrijft een handige benadering voor het integreren van optische vangmethoden en oppervlakte-verbeterde Raman-spectroscopie (SERS) om plasmonische nanodeeltjes te manipuleren voor gevoelige moleculaire detectie. Zonder aggregeringsmiddelen assembleert de vanglaser plasmonische nanodeeltjes om de SERS-signalen van doelanalyten te verbeteren voor in situ spectroscopische metingen.

Optische overvulling van plasmonische nanodeeltjes voor in situ oppervlakte-verbeterde Raman-spectroscopiekarakteriseringen

Hier beschrijven we een snelle magnetische pincetopstelling die nanomechanische metingen uitvoert op krachtgevoelige biomoleculen met de maximale snelheid van 1,2 kHz. We introduceren de toepassing ervan op DNA-haarspelden en SNARE-complexen als modelsystemen, maar het zal ook van toepassing zijn op andere moleculen die betrokken zijn bij mechanobiologische gebeurtenissen.

High-speed magnetisch pincet voor nanomechanische metingen aan krachtgevoelige elementen

Leer plasmonische nanotangentechnieken voor real-time monitoring van ongemodificeerde eiwitten op enkelvoudig molecuulniveau. Deze methode onthult conformatiedynamica relevant voor ziekten zoals Alzheimer en Parkinson.

Monitoring van conformationele dynamica van enkelvoudige ongemodificeerde eiwitten met behulp van plasmonische nanopincetten

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