Electron Microscopic Detection of Proteins and Protein Complexes in Mammalian Cells Using APEX-tagged, Conditionally Stable Nanobodies
利用APEX标记的条件性稳定纳米体电子显微检测哺乳动物细胞中的蛋白和蛋白复合物
(*contributed equally to this work) 发布: 2018年11月20日第8卷第22期 DOI: 10.21769/BioProtoc.3088 浏览次数: 5499
评审: Amey RedkarAnna Maria Ochocka-FoxHolger Andreas Russ
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参见作者原研究论文
The authors used this protocol in:
Apr 2018
Abstract
The recent development of 3D electron microscopic techniques for cells and tissues has necessitated the development of new methods for the detection of proteins and protein-complexes in situ. The development of new genetic tags, such as the ascorbate peroxidase, APEX, for electron microscopic detection of tagged proteins has expanded the available toolbox and ushered in a new era in biological electron microscopy. Here, we describe methods for combining conditionally-stable nanobodies to fluorescent protein tags with APEX-based detection. These methods are compatible with detection of low levels of expression of fluorescently-tagged proteins and with detection of protein complexes using split GFP-based complementation methods. We describe a simple protocol for applying these methods to the electron microscopic detection of proteins and protein complexes in cultured cells.
Keywords: Electron microscopy (电子显微镜)
GFP (GFP)
Nanobodies (纳米抗体)
APEX (APEX)
Peroxidase (过氧化物酶)
Localization (定位)
Background
Recently, we developed a system for the detection of GFP-labeled proteins in transfected cells by electron microscopy (Ariotti et al., 2015). This simple system involves the co-transfection of a plasmid encoding a GFP binding nanobody; a small, genetically encodable, single chain antibody (Kubala et al., 2010), fused to an ascorbate peroxidase APEX2-tag (Martell et al., 2012), which is able to catalyze a reaction to produce a precipitate visible in the electron microscope. More recently, we have expanded the scope of this methodology with i) the addition of an mCherry-binding nanobody allowing detection of mCherry-tagged proteins, ii) the adoption of conditionally stable nanobody sequences and iii) the application to bimolecular fluorescence complementation (Hu et al., 2002), allowing detection of pairwise interactions at the ultrastructural level (Ariotti et al., 2018).
Materials and Reagents
- 9.2 cm2 (40 mm diameter) tissue culture dishes (TPP, supplied by Sigma-Aldrich, catalog number: 93040)
- Gloves
- 0.2 µm syringe filter
- 3 slot grids (Ted Pella, catalog number: 01816) (Figure 1)
Figure 1. 3 slot grids - APEX2-csGBP vector (Addgene, catalog number: 108874) (store at -20 °C)
- mKate2-P2A-APEX2-csGBP vector (Addgene, catalog number: 108875) (store at -20 °C)
- APEX2-csChBP vector (Addgene, catalog number: 108876) (store at -20 °C)
- EGFP-P2A-APEX2-csChBP vector (Addgene, catalog number: 108877) (store at -20 °C)
- mVenus-N vector with insert of choice cloned into EcoRV/SpeI (Addgene, catalog number: 108887) (store at -20 °C)
- mVenus-C vector with insert of choice cloned into EcoRV/SpeI (Addgene, catalog number: 108888) (store at -20 °C)
- Lipofectamine 3000 (Life Technologies, catalog number: L3000015) (store at 4 °C)
- Bovine hemin (Sigma-Aldrich, catalog number: H9039) (store at 4 °C)
- 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich, catalog number: D4168) (store at -20 °C)
- Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16220) (store at 4 °C)
- Hydrogen peroxide (Sigma-Aldrich, catalog number: H1009) (store at 4 °C)
- Osmium tetroxide (OsO4) (ProSciTech, catalog number: C010) (store at 4 °C). Acutely toxic and corrosive.
- LX112 resin kit (Ladd Research, catalog number: 21310) (store at -20 °C when made up)
- Sodium cacodylate trihydrate (Sigma-Aldrich, catalog number: C0250) (store at 4 °C)
- Ethanol (Chem-supply, catalog number: EA043-2.5L-P)
- Hydrochloric Acid 32% (RCI, catalog number: RP1104)
- NaOH
- KCl
- Na2HPO4
- KH2PO4
- NaCl
- Bovine hemin (if necessary) (see Recipes)
- PBS, pH 7.4 (see Recipes)
- 2.5% glutaraldehyde in 0.1 M cacodylate buffer (see Recipes)
- 0.2 M cacodylate buffer (see Recipes)
- DAB/Cacodylate wash mixture and DAB/cacodylate reaction mixture (see Recipes)
- Osmium tetroxide solution (see Recipes)
- Ethanol series for dehydration (see Recipes)
- LX112 resin ethanol series (see Recipes)
Equipment
- Pliers (JBS, catalog number: 00718811) or similar
- Oven (SEM, model: 24) or similar
- PELCO Biowave with PELCO ColdSpot Pro system for temperature control (Ted Pella) (Figure 2)
Figure 2. PELCO Biowave - JEM-1011 Transmission Electron Microscope (JEOL) or similar
- Leica EM UC6 Ultramicrotome (Leica Microsystems) or similar
- Morada CCD camera (Olympus Soft-Imaging-Solutions) or similar (Figure 3)
Figure 3. JEM-1011 electron microscope (left), Leica EM U6 Ultramicrotome (middle), Morada CCD camera (right) - 45° diamond knife (Diatome, supplied by Agar Scientific, catalog number: AGG339-5) (Figure 4)
Figure 4. 45° diamond knife - Project specific tissue culture equipment such as a class II biological safety cabinet, CO2 incubator etc.
- Fume hood
- Vortex
Software
- iTEM (EMSIS GmbH) v5.2, build 4768, license required, https://www.emsis.eu/products/software/item
- Photoshop CC (Adobe), license required, https://www.adobe.com/au/creativecloud.html
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Hall, T. E., Rae, J., Ariotti, N. and Parton, R. G. (2018). Electron Microscopic Detection of Proteins and Protein Complexes in Mammalian Cells Using APEX-tagged, Conditionally Stable Nanobodies. Bio-protocol 8(22): e3088. DOI: 10.21769/BioProtoc.3088.
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分类
发育生物学 > 形态建成 > 细胞结构
细胞生物学 > 细胞成像 > 电子显微镜
细胞生物学 > 细胞成像 > 固定组织成像
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