使用秀丽隐杆线虫裂解液检测和定量聚谷氨酰胺聚集体的滤器延迟试验

Olga Sin; Alejandro Mata-Cabana; Renée I. Seinstra; Ellen A. A. Nollen

doi:10.21769/BioProtoc.3042

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates

使用秀丽隐杆线虫裂解液检测和定量聚谷氨酰胺聚集体的滤器延迟试验

Olga Sin

AM Alejandro Mata-Cabana

RS Renée I. Seinstra

EN Ellen A. A. Nollen email

发布: 2018年10月05日第8卷第19期 DOI: 10.21769/BioProtoc.3042 浏览次数: 8274

评审: Saumik BasuManoj B. MenonAnonymous reviewer(s)

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Molecular Cell, featuring study using the protocol.

Mar 2017

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

秀丽隐杆线虫中细胞核/细胞质蛋白的分离

Alejandro Mata-Cabana [...] Ellen A. A. Nollen

2018年10月20日 9888 阅读

具有亚细胞空间分辨率的蛋白质周转动态分析

Lorena Alamillo [...] Edward Lau

2025年08月05日 897 阅读

测定丙酮酸激酶动力学的优化酶偶联分光光度法

Saurabh Upadhyay

2025年08月20日 838 阅读

Abstract

Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein’s native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a C. elegans model of polyglutamine diseases, we describe in detail the filter retardation assay, a method that captures protein aggregates in a cellulose acetate membrane and allows its detection and quantification by immunoblotting.

Keywords: Protein aggregation (蛋白质聚集)

Polyglutamine (多聚谷氨酰胺)

Amyloid (淀粉样蛋白)

Filter trap assay (过滤捕集试验)

Filter retardation assay (过滤阻滞测定)

Dot blot (斑点印迹)

C. elegans (秀丽隐杆线虫)

Background

One pathological feature of neurodegenerative diseases like Parkinson’s, Alzheimer’s and polyglutamine diseases is the presence of protein aggregates in distinct areas of the brain (reviewed in Soto, 2003; Stroo et al., 2017). In the case of polyglutamine diseases, abnormal expansion of glutamine (CAG) repeats in the coding sequence disturbs the native folding of the protein. As a result, the misfolded protein exposes regions of its amino acid sequence, which makes it prone to aggregate with other proteins, forming large, insoluble aggregates that can hamper normal cellular function (reviewed in Kuiper et al., 2017).

Several methods have been developed for the detection of insoluble protein aggregates including, for instance, dye binding assays (e.g., Thioflavin T, Congo red, NIAD-4) and electron microscopy. Filter retardation assay is a quick and sensitive method that detects and quantifies protein aggregates formed in vivo and in vitro, including polyglutamine (Scherzinger et al., 1997; Wanker et al., 1999), alpha-synuclein (Recasens et al., 2018), and amyloid-beta aggregates (Bieschke et al., 2009). In this assay, sodium dodecyl sulfate (SDS)-resistant protein aggregates are filtered and retained in a cellulose acetate membrane, while monomeric intermediate species are not captured. The protein aggregates retained in the membrane are subsequently detected by antibodies, which allows for their quantification.

This protocol describes a method to detect and quantify SDS-resistant polyglutamine aggregates in the nematode Caenorhabditis elegans and can be applied to investigate the aggregation of aggregation-prone proteins in vivo and in vitro.

Materials and Reagents

200 µl pipette tips (Greiner Bio-one, catalog number: 741065 )
1000 µl pipette tips (Greiner Bio-one, catalog number: 741045 )
15 ml conical tubes (SARSTEDT, catalog number: 62.554.502 )
2 ml Screw cap tubes (SARSTEDT, catalog number: 72.693.105 )
0.5 mm Glass beads (Carl Roth, catalog number: N030.1 )
11.3 x 7.7 cm Bio-Dot/Bio-Dot SF Filter Paper (Bio-Rad Laboratories, catalog number: 1620161 )
Cellulose acetate membranes, 0.22 micron, Sphaero (Sterlitech, catalog number: CA023001 )
1.5 ml microfuge tube (Greiner Bio One International, catalog number: 616201 )
Rubber seal
Plastic wrap
50 ml conical tube (SARSTEDT, catalog number: 62.547.004 )
Worms
Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23227 )
Complete protease inhibitors (Roche Diagnostics, catalog number: 11697498001 )
ECL^TM Prime Western Blotting Detection (GE Healthcare, Amersham, catalog number: RPN2232 )
Liquid nitrogen
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
1 M Tris-HCl
5 M NaCl
10% SDS
Milli-Q water
Non-fat dry milk powder (Campina)

Antibodies against green fluorescent protein (GFP)/yellow fluorescent protein (YFP) and tubulin (Table 1)

Table 1. List of primary and secondary antibodies

Primary antibody	Host	Company/catalog number	Dilution primary antibody	Secondary antibody
α-GFP	Mouse	TaKaRa Bio, Clontech Laboratories ( 632381 )	1:5,000	Anti-mouse (1:10,000) Bio-Rad ( 1706516 )
α-tubulin	Mouse	Sigma-Aldrich ( T6074 )	1:5,000	Anti-mouse (1:10,000) Bio-Rad ( 1706516 )

Sodium phosphate dibasic heptahydrate (Na₂HPO₄•7H₂O) (Acros Organics, catalog number: 424380010 )
Sodium chloride (NaCl) (Merck, catalog number: 1064041000 )
Potassium chloride (KCl) (Fisher Chemicals, catalog number: 10010310 )
10x Phosphate buffered saline (PBS) (see Recipes)
1x PBS-Triton 0.1% (see Recipes)
1x PBS-Triton 0.05% (see Recipes)
Filter trap assay (FTA) sample buffer (see Recipes)
FTA wash buffer (see Recipes)
1 M dithiothreitol (DTT, see Recipes)
Blocking solution (see Recipes)

Equipment

P20 Pipetman (Gilson, catalog number: F123600 )
P200 Pipetman (Gilson, catalog number: F123601 )
P1000 Pipetman (Gilson, catalog number: F123602 )
30-300 μl multi-channel pipette (Eppendorf, catalog number: 3125000052 )
10 ml Serological glass pipettes (SARSTEDT, catalog number: 86.1254.001 )
Centrifuge (Thermo Fisher Scientific, model: SL 40R , rotor: TX-750 )
Bead beater (MP Biomedicals, model: FastPrep-24 )
48-well Bio-Dot Microfiltration System (Bio-Rad Laboratories, catalog number: 1703938 )
Vacuum pump
20 °C incubator (LIEBHERR, model: WK 4126 )
Vortex (Fisher scientific, model: ZX Wizard vortex, catalog number: 11746744 )
Fuji Super RX-N 13x18 film (Fujifilm, catalog number: RX1318 )
Autoclave

Software

ImageJ (Open source: https://imagej.nih.gov/ij/)
Microsoft Excel (Microsoft Corporation, Redmond, USA)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Sin, O., Mata-Cabana, A., Seinstra, R. I. and Nollen, E. A. A. (2018). Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates. Bio-protocol 8(19): e3042. DOI: 10.21769/BioProtoc.3042.