Atomic force mikroskopi Imaging og Kraft spektroskopi af understøttede lipiddobbeltlag
Summary
We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.
Abstract
Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force.
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.
Introduction
Atomic force mikroskopi (AFM) genererer et billede af en overflade ved at scanne igennem et område af prøven under anvendelse af en udligger med en meget skarp spids 1. Bevægelsen af cantilever sonder overflade topologi af prøven. AFM har været almindeligt anvendt på biologiske molekyler – herunder proteiner, DNA, og membraner, på grund af sin alsidighed i at analysere faste prøver i luften eller næsten native tilstand i flydende 2-5.
Bortset fra dens høj opløsning billeddannelse kapacitet i nanometerområdet, AFM cantilever virker som en fjeder til at probe interaktion styrker (adhæsions- og frastødning) og mekaniske egenskaber ved prøve 5,6. Dette er kendt som force spektroskopi. I denne tilstand sonden først nærmer prøven og udøver en kraft på det, så trækkes tilbage, indtil den mister kontakt med prøven (figur 1A). De genererede kurver viser kraft som funktion af afstand af cantilever for både appskalle og tilbagetrækning. Adskillige egenskaber, herunder elasticitetsmodulet for at måle stivheden af et materiale, og adhæsionskræfter kan afledes.
Understøttede lipiddobbeltlag er biologiske modelmembraner liggende på toppen af en fast bærer – som regel glimmer, borsilicatglas, smeltet silica, eller oxideret silicium 7. De fremstilles under anvendelse af forskellige teknikker som vesikel deposition, Langmuir-Blodgett fremgangsmåde og spin-coating af 8,9. AFM billeddannelse er blevet anvendt til at følge dannelsen af disse understøttede dobbeltlag 10, og sonde forskellige strukturer dannet af membraner af forskellige sammensætninger 11-15.
Udførelse kraft spektroskopi på understøttede dobbeltlag resulterer i en top i tilgang kurve. Denne top angiver den nødvendige kraft til at gennembore dobbeltlaget, og kaldes gennembrud kraft. Dobbeltlaget Tykkelsen kan også måles ved hjælp af kraft kurve 6. Den typiske gennembrud kraft dobbeltlagområdet mellem 1-50 nN 6. Disse egenskaber afhænger af lipid pakning (flydende eller gel-fase) og struktur (acylkædelængde og grad af umættethed) og ændret af membran-midler 16. Teorien bag brud er blevet forklaret 17 og andre eksperimentelle parametre såsom cantilever blødhed, tip radius og tilgang hastighed påvirker også gennembruddet force 15,16,18. Kraft spektroskopi er blevet anvendt til at analysere egenskaber af forskellige lipidfaser 11,19, sammensætning-afhængige ændringer 12,20, samt virkningerne af andre biomolekyler, lignende peptider, på stabiliteten af membranen 21.
Den flade orientering af understøttede dobbeltlag er fordelagtig til at kombinere AFM med andre metoder såsom overfladeplasmonresonans 22 og fluorescensmikroskopi 11,19 til bedre karakterisere struktur og egenskaber af membraner.
Denne detaljerede video protocol er beregnet til at forberede understøttede lipiddobbeltlag hjælp vesikel deposition og analysere dem med AFM og force spektroskopi. Mens vesikler af forskellige størrelser kan anvendes til fremstilling af dobbeltlag, denne protokol fokuserer på små og store unilamellare vesikler. Understøttede dobbeltlag denne fase adskille i væske bestilt (L o) og flydende uordnede (L d) faser blev karakteriseret 11,15. Membranen består af di-oleoyl-phosphatidylcholin (DOPC), sphingomyelin (SM) og cholesterol (Chol) ved 2: 2: 1 forhold. Denne sammensætning modeller lipidklumperne, som foreslås at opføre sig som platforme vigtige for protein handel og sortering, cellesignalering og andre cellulære processer 23,24.
Protocol
Representative Results
Discussion
SLBs sammensat af DOPC: SM: Chol (2: 2: 1) blev dannet på glimmer efter vesikel adsorption og brud induceret af calciumchlorid. Denne lipidsammensætning adskilt i L D og L o faser. L o fase beriges med sphingomyelin og cholesterol og er mindre fluid / mere tyktflydende (figur 1A) end L d fase 11. Adskillelsen af L o fra L d fase manifesterer sig som cirkulære strukturer hævet over det omgivende (figur 1B, C).</stron…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af Max Planck Society, det tyske Cancer Research Center, University of Tübingen, og Bundesministerium für Bildung und Forschung (bevilge nr. 0312040).
Vi takker Eduard Hermann for at hjælpe os automatisere analysen af den kraft kurven data og Dr. Jakob Suckale for nærlæsning af dette manuskript.
Materials
| 1,2-dioleoyl-sn-glycero-3-phosphocholine | Avanti Polar Lipids, Inc. | 850375P | Comes as lyophilized powder in sealed vials. Dissolve all powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20°C. For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&view=article&id=1679&Itemid=398 |
| Sphingomyelin (Brain, Porcine) | Avanti Polar Lipids, Inc. | 860062P | Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20°C. For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&view=article&id=1679&Itemid=398 |
| Cholesterol | Avanti Polar Lipids, Inc. | 700000P | Comes as lyophilized powder in large sealed plastic containers. Dissolve a spatula point of powder powder in chloroform upon opening. Store extra as dried lipid films, under inert atmosphere, at -20°C. For more information on storage and handling visit http://www.avantilipids.com/index.php?option=com_content&view=article&id=1679&Itemid=398 |
| Sodium chloride (NaCl), 99.8% | Carl Roth GmbH + Co. KG | 9265.1 | |
| Potassium chloride (KCl), 88% | Sigma | P9541 | |
| Sodium hydrogenphosphate (Na2HPO4), >99% | AppliChem GmbH | A1046 | |
| Potassium dihydrogenphosphate (KH2PO4), 99% | Carl Roth GmbH + Co. KG | 3904.1 | |
| Calcium chloride dihydrate (CaCl2), molecular biology grade | AppliChem GmbH | A4689 | |
| HEPES, molecular biology grade | AppliChem GmbH | A3724 | |
| Glass coverslip, 24×60 mm, 1mm thickness | Duran Group | 2355036 | |
| Mica blocks | NSC Mica Exports Ltd. | These are mica pieces at least 1 sq. Inches in area and thickness randing from 0.006 inches to 0.016 inches. They are cut to a specific size by the company for shipping. Small mica discs can be punched from the mica blocks using the punch and die set. Always handle mica with gloves or tweezers. | |
| Punch and Die Set | Precision Brand Products, Inc | 40105 | |
| Optical Adhesive | Norland Products, Inc. | NOA 88 | Liquid adhesive that hardens when cured under long wavelength UV light. |
| Laboratory Equipment Grease | Borer Chemie AG | Glisseal N | |
| Liposome Extruder | Avestin | LiposoFast-Basic | As an alternative one can also look at offers from Northern Lipids, Inc. |
| Adhesive Tape | 3M | Scotch(R) Magic (TM) Tape 810 (1-inch) | |
| Bath Sonicator | Bandelin Sonorex Digitec | DT-31 | No heating, Frequency: 35 kHz, Ultrasonic Peak Output: 160 W, HF Power: 40 W. http://www.bandelin.com/datenblaetter/dt/DT_31_H_1798d_DE_GB_FR_BANDELIN.pdf |
| Silicon Nitride AFM Cantilever | Bruker AFM Probes | DNP-10 | Each cantilever has four tips and their nominal tip radius is 20 nm (with possible maximum at 60 nm). Based on the specifications, we use tip D with resonance frequency of 18 kHz, and nominal spring constant of 0.06 N/m. |
| AFM | JPK | JPK Nanowizard II mounted on Zeiss Axiovert 200 |
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