密度梯度离心法富集和鉴定丝状真菌中GFP标记的壳聚糖小囊泡

Jorge Verdín; Eddy Sánchez-León; Rosa Fajardo-Somera; Carlos A. Leal Morales; Salomón Bartnicki-García; Meritxell Riquelme

doi:10.21769/BioProtoc.1611

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Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi

密度梯度离心法富集和鉴定丝状真菌中GFP标记的壳聚糖小囊泡

Jorge Verdín ^*

Eddy Sánchez-León ^*

RF Rosa Fajardo-Somera

CM Carlos A. Leal Morales

SB Salomón Bartnicki-García

MR Meritxell Riquelme email

(*contributed equally to this work) 发布: 2015年10月05日第5卷第19期 DOI: 10.21769/BioProtoc.1611 浏览次数: 15302

评审: Fanglian HeAksiniya AsenovaSadri Znaidi

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Cover of Molecular Microbiology, featuring study using the protocol.

Feb 2015

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Abstract

Density gradient centrifugation has been utilized to characterize the subcellular distribution of physiologically relevant enzymes in yeasts and filamentous fungi (Leal-Morales et al., 1988; Martínez et al., 1989; Kamada et al., 1991). This approach is now potentiated by protein tagging and live imaging techniques, which make possible to relate a single protein with, for example, a discrete population of intracellular vesicles and their in vivo dynamics (Verdín et al., 2009; Fajardo-Somera et al., 2013; Sánchez-León et al., 2015). Here, we describe the density gradient centrifugation and fractionation analysis of cell-free homogenates of a Neurospora crassa (N. crassa) strain that expresses CHS-6 chitin synthase fused to the green fluorescent protein (Riquelme et al., 2007).

Keywords: Neurospora crassa (粗糙脉孢菌)

Chitosomes (chitosomes)

Vesicles (囊泡)

Fractionation (分馏)

Materials and Reagents

Tygon R-3603 tubing, I.D. 1/16 inch (Saint-Gobain, catalog number: AAC00002 )
Grade 1 Qualitative Filter Papers (GE Healthcare, catalog number: 1001-090 )
Neurospora crassa conidia
Sucrose (Sigma-Aldrich, catalog number: S0389 )
Sodium phosphate monobasic (NaH₂PO₄^.H₂O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S369-500 )
Sodium phosphate dibasic anhydrous (Na₂HPO₄) (Sigma-Aldrich, catalog number: S-7907 )
Protease Inhibitor Cocktail, Complete ULTRA tablets EDTA-free (Roche Diagnostics, catalog number: 04693132001 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 04693132001 ”.
Glass beads (500 μm) (Bio Spec Product, catalog number: 11079105 )
10% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
65% (w/v) sucrose in 33 mM (final) phosphate buffer (pH 8.2) (steam sterilize and store at 4 °C until use)
D-Biotin (Faga Labs, catalog number: CAS-58-85-5 )
Vogel’s complete medium (see Recipes)
50x salt solution (see Recipes)
Trace element solution (see Recipes)
100 mM phosphate buffer (pH 8.2) (see Recipes)
Laemmli buffer (see Recipes)

Equipment

Braun MSK 50 ml shaking bottles (LABEQUIP LTD, catalog number: 8541302 )
VWR^® Standard Hot Plate Stirrers (VWR International, catalog number: 12365-382 )
Quick-seal centrifuge tube (Beckman Coulter, catalog number: 344326 )
Shaker incubator (Lab-LineX, model: Orbit Environ Shaker )
Vacuum filtration system
Braun MSK cell homogenizer (LABEQUIP LTD, catalog number: 953030 )
Bright field microscope (Olympus, model: Vanox-S )
Beckman L8-70M Ultracentrifuge (pre-cooled at 4 °C) (Beckman Coulter)
Type 70Ti rotor (pre-cooled at 4 °C) (Beckman Coulter)
GM-40 Linear Gradient maker (C. B. S. Scientific)
Peristaltic (Multi-staltic) pump (Buchler Lab, catalog number: N/A )
Tube sealer (Beckman Coulter, catalog number: 342420 )
Density gradient fractionator (ISCO, 185)
Note: This model is not available anymore. Brandel BR-186 Gradient Fractionator with Syringe Pump can be used instead.
CIGNET^TM Fraction collector (ISCO)
Note: This item is also discontinued, but Bio-Rad 2110 Fraction Collector can be used instead.
6505 W UV-Vis Spectrophotometer (Bibby-scientific, Jenway)
Refractometer (ZEISS, catalog number: 12230 )

Procedure

English

中文翻译

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如何引用

Verdín, J., Sánchez-León, E., Fajardo-Somera, R., Morales, C. A. L., Bartnicki-García, S. and Riquelme, M. (2015). Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi. Bio-protocol 5(19): e1611. DOI: 10.21769/BioProtoc.1611.