Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation
富含精氨酸的肽可以主动介导液-液相分离
发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2525 浏览次数: 15033
评审: Nicoletta CordaniWeiyan JiaMirko Messa
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参见作者原研究论文
The authors used this protocol in:
Mar 2017
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Abstract
Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems et al., 2017).
Keywords: LLPS (LLPS)
Amyotrophic lateral sclerosis (肌萎缩性侧索硬化)
Peptides (肽)
Arginine (精氨酸)
Phase separation (相分离)
Mass spec (质谱仪)
C9orf72 (C9orf72)
Stress granule (应激颗粒)
Background
Arginine-rich disordered domains are often found in RNA binding proteins, including the ones associated with neurodegenerative diseases (e.g., FUS, FMRP, hnRNPA1) (Varadi et al., 2015; Boeynaems et al., 2017). Also toxic arginine-rich repeat peptides (i.e., polyGR and polyPR) are produced in amyotrophic lateral sclerosis patients carrying the C9orf72 repeat expansion (Kwon et al., 2014; Mizielinska et al., 2014; Varadi et al., 2015; Boeynaems et al., 2016). While phase separation of uncharged low complexity domains had been studied before (Kato et al., 2012; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Patel et al., 2015), we developed this protocol to test whether arginine-rich domains could also contribute to phase separation. A schematic representation of the protocol can be seen in Figure 1.
Figure 1. Workflow of protein droplet formation and analysis. A diffuse arginine-rich peptide solution can be induced to undergo liquid-liquid phase separation by addition of RNA or molecular crowder (PEG). Resulting droplets can be further analyzed by fluorescence microscopy and FRAP.
Materials and Reagents
- Pipette tips
- Amicon® Ultra–0.5 ml centrifugal filters 3K (Merck, catalog number: UFC500396 )
- Cell counting slides (Bio-Rad Laboratories, catalog number: 1450015 )
- Clear adhesive tape
- Custom 20 to 60 amino acid peptides with free carboxy and amino-termini (Pepscan)
- Custom Alexa labeled RNA 30 ribonucleotide oligomers (IDT)
- MilliQ water
- Alexa Fluor® Protein Labeling Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: A10235 )
- PEG300 (Sigma-Aldrich, catalog number: 81160 )
Note: This product has been discontinued. - Potassium phosphate monobasic (KH2PO4)
- Potassium phosphate dibasic (K2HPO4)
- Polyuridylic acid potassium salt (Sigma-Aldrich, catalog number: P9528 )
- Clear nail varnish
- 10x potassium buffer, pH 7 (see Recipes)
Equipment
- Pipette
- Bench-top centrifuge (Eppendorf, models: 5430 and 5810 R )
- trUView cuvettes (Bio-Rad Laboratories, catalog number: 1702510 )
Note: This product has been discontinued. - SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, model: SmartSpec Plus )
- LSM 780 Meta NLO confocal microscope (ZEISS, model: LSM 780 NLO ) with 20x long range objective (ZEISS, model: LD Plan-Neofluor 20x/0.4 Corr Ph2 M27, catalog number: 421351-9970-000 )
Software
- Zen software (ZEISS)
- GraphPad Prism (GraphPad)
- Excel (Microsoft)
Procedure
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文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Boeynaems, S., De Decker, M., Tompa, P. and Van Den Bosch, L. (2017). Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation. Bio-protocol 7(17): e2525. DOI: 10.21769/BioProtoc.2525.
Download Citation in RIS Format
分类
神经科学 > 神经系统疾病 > 血脑屏障
生物化学 > 蛋白质 > 分离和纯化
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