シングルビリオン原子間力顕微鏡や超解像蛍光イメージングのためのサンプル調製

1Department of Physics & Astronomy, University of Utah, 2Center for Cell and Genome, University of Utah
Published 1/02/2014
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Hodges, J. A., Saffarian, S. Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging. J. Vis. Exp. (83), e51366, doi:10.3791/51366 (2014).

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Abstract

ガラス表面へのビリオンの固定化は、単一ビリオンイメージングにおいて重要なステップである。ここでは、特異性でガラス表面に単一のビリオンの付着を可能にする単一分子イメージングアッセイから採用された技術を提示する。この調製物を、アビジンの層を追加し、最後に、ビオチン化ウイルス特異的抗体の結合を介してビリオンアンカーを作成し、PLL-gのPEG-PLL-及びγ-PEG-ビオチンの混合物を用いてガラスの表面をグラフト化に基づいている。我々は、原子間力顕微鏡(AFM)および超解像蛍光イメージングを含めた実験の範囲にわたってこの技術を適用している。この試料調製方法は、表面へのビリオンの制御密着性が得られる。

Materials

Name Company Catalog Number Comments
Glass coverslips (fPALM) Electron Microscopy Services 72225-01 25 mm Coverslips
PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)  SuSoS - Stock: 0.5 mg/ml in PBS
NeutrAvidin biotin-binding protein Invitrogen A2666 Stock: 0.25 mg/ml in PBS
Alexa Fluor 647 goat anti-rabbit IgG (H+L)  Invitrogen A21245 1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer
α-VSV-G  Invitrogen ab34774 1:200 in 0.2 M KPO4, 0.15 M NaCl, 10% Glycol, pH 7.2 buffer
Gluox Sigma G2133-250KU Glucose oxidase type seven from Aspergillius 
Catalase Sigma C40-100mg Catalase from Bovine liver 
MEA Sigma 30070-10G Cysteamine (MEA
Stock Buffer 50 mM Tris-HCl (pH 8.0) + 10 mM NaCl + 10% glucose
NTE 10 mM Tris pH 7.4, 100 mM NaCl, 66 mM EDTA
Slide-A-Lyzer, mini dialysis unit Thermo Scientific 10,000 MW
Microscope Cover Glass: 35 CIRCLE #1 Fisherbrand 35 CIRCLE #1

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