采用荧光漂白恢复法（FRAP）评估蛋白质合成速率

Nikos  Kourtis; Nektarios  Tavernarakis

doi:10.21769/BioProtoc.2156

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Peer-reviewed

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

采用荧光漂白恢复法（FRAP）评估蛋白质合成速率

NK Nikos Kourtis

Nektarios Tavernarakis email

发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2156 浏览次数: 12255

评审: Jyotiska ChaudhuriVarpu MarjomakiAnonymous reviewer(s)

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Abstract

Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

Keywords: Caenorhabditis elegans (秀丽隐杆线虫)

FRAP (荧光漂白恢复)

Messenger RNA (信使RNA)

Protein synthesis (蛋白质合成)

Protein translation (蛋白质翻译)

Background

Proper regulation of protein synthesis is critical for cell homeostasis and growth. Deregulation of protein synthesis has been implicated in pathologies such as cancer and senescent decline (Bjornsti and Houghton, 2004; Syntichaki et al., 2007). Currently available biochemical methods for measuring general protein synthesis rate include metabolic labeling and polysomal profiling (Martin, 1998; Rennie et al., 1994). The applicability of these methodologies is limited due to poor intake and uncontrolled or unequal distribution of the label throughout the animal or tissue of interest. Also, these methods lack specificity and significant changes in specific cells or tissues of interest may be masked due to variability in intrinsic rates of translation of the bulk of the sample. In this protocol, we describe a method for monitoring protein synthesis rates in the nematode Caenorhabditis elegans, based on fluorescence recovery after photobleaching (FRAP). The experimental approach is based on the expression of fluorescent proteins, in cells and tissues of interest of transgenic animals. Fluorescence is then photobleached by irradiating cells, tissues or whole animals with a powerful light source. Recovery of fluorescence, indicative of new protein synthesis, is then monitored in cells or tissues of interest.

Materials and Reagents

Greiner Petri dishes (60 x 15 mm) (Greiner Bio One, catalog number: 628161 )
35 mm plates (Corning, catalog number: 430165 )
Microscope slides 75 x 25 x 1 mm (Marienfeld-Superior, catalog number: 10 006 12 )
Microscope cover glass 18 x 18 mm (Marienfeld-Superior, catalog number: 01 010 30 )
C. elegans strains (wild type [N2], ife-2[ok306], N2; Ex[p_ife-2GFP, pRF4], ife-2[ok306]; Ex[p_ife-2GFP, pRF4])
Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
Cycloheximide (Sigma-Aldrich, catalog number: C7698 )
Note: Cycloheximide has significant, toxic side effects.
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: 7778-77-0 )
Potassium phosphate dibasic (K₂HPO₄) (Sigma-Aldrich, catalog number: 7758-11-4 )
Sodium chloride (NaCl) (EMD Millipore, catalog number: 106404 )
Peptone (BD, Bacto, catalog number: 211677 )
Streptomycin (Sigma-Aldrich, catalog number: S6501 )
Agar (Sigma-Aldrich, catalog number: 05040 )
Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C5080 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M7506 )
Nystatin stock solution (Sigma-Aldrich, catalog number: N3503 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: 7558-79-4 )
Phosphate buffer (1 M; sterile, see Recipes)
Nematode growth medium (NGM) agar plates (see Recipes)
M9 buffer (see Recipes)

Equipment

Dissecting stereomicroscope (Nikon, model: SMZ645 )
UV crosslinker (VilberLourmat, model: BIO-LINK – BLX-E365 )
Epifluorescence microscope (ZEISS, model: Axioskop 2 Plus )
Standard equipment for preparing agar plates (autoclave, Petri dishes, etc.) (Sambrook and Russell, 2001)
Standard equipment for maintaining worms (platinum wire pick, incubators, etc.)
Note: For basic C. elegans culture, maintenance and manipulation techniques see refs (Epstein and Shakes, 1995; Lewis and Fleming, 1995; Hope, 1999; Strange, 2006). For information on C. elegans biology see refs (Wood, 1988; Epstein and Shakes, 1995; Riddle, 1997) and WormBook (http://www.wormbook.org/).

Software

Camera control and imaging software (Carl Zeiss, Axio Vision 3.1 software)
ImageJ image processing software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/) (Abràmoff et al., 2004)
Microsoft Office 2011 Excel software package (Microsoft Corporation, Redmond, USA)
Prism software package (GraphPad Software Inc., San Diego, USA)

Procedure

English

中文翻译

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如何引用

Kourtis, N. and Tavernarakis, N. (2017). Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP). Bio-protocol 7(5): e2156. DOI: 10.21769/BioProtoc.2156.