啮齿动物体外培养神经元的细胞膜蛋白质的生物素化和纯化

Margarida Caldeira; Joana S. Ferreira; Ana Luísa Carvalho; Carlos B. Duarte

doi:10.21769/BioProtoc.1807

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Peer-reviewed

Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons

啮齿动物体外培养神经元的细胞膜蛋白质的生物素化和纯化

MC Margarida Caldeira

JF Joana S. Ferreira

AC Ana Luísa Carvalho

Carlos B. Duarte email

发布: 2016年05月20日第6卷第10期 DOI: 10.21769/BioProtoc.1807 浏览次数: 10286

评审: Oneil G. BhalalaPamela MaherEdgar Soria-Gomez

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Jun 2015

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Abstract

This protocol aims at the biotin labeling and affinity purification of plasma membrane proteins from cultured neurons. Protein biotinylation consists in the covalent attachment of biotin to proteins. Biotin is a membrane unpermeable molecule with a small size (MW 244.31 g/mol) and therefore does not interfere with the normal function of proteins. Biotin binds to streptavidin and avidin molecules with high affinity. This binding is extremely resistant to temperature, pH and proteolysis, which allows capture and purification of plasma membrane proteins. Moreover, proteins can bind several biotin molecules, that will allow the consequent binding of several streptavidin or avidin molecules, increasing the sensitivity of detection of the proteins of interest. In this protocol proteins at the cell surface of live cultured neurons are biotinylated. Neuronal extracts are prepared and biotinylated proteins are collected with NeutrAvidin-coupled beads, and analyzed by Western blotting.

Keywords: Plasma-membrane proteins (质膜蛋白质)

Affinity purification (亲和纯化)

Biotin (生物素)

Cell surface (细胞表面)

Cultured neurons (培养的神经元)

Materials and Reagents

Centrifuge microtube with 0.45 μm filter (VWR International, catalog number: 82031-360 )
Cell scraper with 1.7 cm blade length (SARSTEDT AG & Co, catalog number: 831830 )
Primary cultures of rodent hippocampal or cortical neurons (as described in Caldeira et al., 2007a; Caldeira et al., 2007b; Ferreira et al., 2015)
Glycine (Sigma-Aldrich, catalog number: 15527 )
NeutrAvidin plus ultralink resin (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 53151 )
EZ link sulfo-nhs-ss-biotin (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 21331 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626-1 g )
Chymostatin (Sigma-Aldrich, catalog number: C7268-1 mg )
Leupeptin (Sigma-Aldrich, catalog number: L2884-1 mg )
Antipain dihydrochloride from microbial source (Sigma-Aldrich, catalog number: A6191-1 mg )
Pepstatin A (Sigma-Aldrich, catalog number: P5318-5 mg )
Deoxycholic acid (DOC) (Sigma-Aldrich, catalog number: D5670 )
Note: it is also named “Sodium deoxycholate monohydrate” on Sigma-Aldrich website.
Dithiothreitol (DTT) (NZYTech, catalog number: MB03101 )
Liquid Nitrogen
DMSO (Sigma-Aldrich, catalog number: D8418-100 ml )
Pierce^TM Bicinchoninic acid protein assay-Reagent A (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23223 )
Pierce^TM Bicinchoninic acid protein assay-Reagent B (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23224 )
NaCl
KCl
KH₂PO₄
Na₂HPO₄
MgCl₂
CaCl₂
Tris-HCl
EGTA
EDTA
Triton X-100
SDS
Sodium orthovanadate
Bromophenol blue
PBS (see Recipes)
PBS/Ca²⁺/Mg²⁺ (see Recipes)
Radioimmune precipitation assay (RIPA) buffer (see Recipes)
Lysis buffer for insoluble proteins (see Recipes)
2x denaturating buffer (see Recipes)
Protease inhibitor cocktail (see Recipes)

Equipment

Orbital horizontal shaker (GFL, model: 3005 )
Sonicator (Sonics & Materials, model: VC50 )
Benchtop microcentrifuge (Eppendorf AG, model: 5415D )
Laboratory tube rotator (Falc Intruments s.r.l., model: F205 )
Heating block (Bibby Scientific, Stuart Scientific, model: SHT200D )

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Caldeira, M., Ferreira, J. S., Carvalho, A. L. and Duarte, C. B. (2016). Biotinylation and Purification of Plasma Membrane-associated Proteins from Rodent Cultured Neurons. Bio-protocol 6(10): e1807. DOI: 10.21769/BioProtoc.1807.
Ferreira, J. S., Schmidt, J., Rio, P., Águas, R., Rooyakkers, A., Li, K. W., Smit, A. B., Craig, A. M. and Carvalho, A. L. (2015). GluN2B-containing NMDA receptors regulate AMPA receptor traffic through anchoring of the synaptic proteasome. J Neurosci 35(22): 8462-8479.