Synaptoneurosome Preparation from C57BL/6 Striata
从C57BL/6 小鼠纹状体制备突触神经小体
发布: 2016年02月20日第6卷第4期 DOI: 10.21769/BioProtoc.1735 浏览次数: 13279
评审: Oneil G. BhalalaAlka MehraMartin V Kolev
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
Abstract
Activity-dependent local mRNA translation endows synapses to remodel their structure and function (Bramham and Wells, 2007). This process is tightly controlled by the state of phosphorylation of several components of the translational machinery including initiation factors and ribosomal proteins (Buffington et al., 2014). The present protocol describes a method to prepare striatal synaptoneurosomes, from adult mice, containing both pre- and postsynaptic elements in which the level of synaptic phospho-proteins can be quantified (Biever et al., 2015).
Keywords: Synaptoneurosome (突触神经小体)
Striatum (纹状体)
Phospho-protein (磷酸化蛋白)
Materials and Reagents
- 1 ml dounce tissue grinder (Capitol scientific, Wheaton®, catalog number: 357538 )
- 5 ml and 60 ml syringes (BD, Plastipak, catalog number: 309647 and 300866 , respectively)
- Nylon net filters 100 μm (Merck Millipore Corporation, catalog number: NY1H02500 )
- Mitex membrane filter 10 μm (Merck Millipore Corporation, catalog number: LCWP02500 )
- 1.5 ml Eppendorf tubes (Eppendorf, catalog number: 00 30120086 )
- C57BL/6 mice (≥8 weeks old, male or female) (Mus musculus)
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
- Potassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P3786 )
- Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: 71630 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
- D-(+)-Glucose (Sigma-Aldrich, catalog number: 67528 / G6728 )
- HEPES (pH 7.4) (Sigma-Aldrich, catalog number: H3375 )
- Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
- Sodium fluorate (Sigma-Aldrich, catalog number: S7920 )
- Sodium pyrophosphate decahydrate (Sigma-Aldrich, catalog number: 221368 )
- Glycerol phosphate disodium salt hydrate (Sigma-Aldrich, catalog number: G6501 )
- Aprotinin (Sigma-Aldrich, catalog number: A1153 )
- Leupeptin hydrochloride (Sigma-Aldrich, catalog number: L0649 )
- Pepstatin (Sigma-Aldrich, catalog number: P4265 )
- Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: 78830 )
- Phospho-S845-GluR1 [Anti-phospho-GluR1 (Ser845) Antibody, clone EPR2148, rabbit monoclonal] (Merck Millipore Corporation, catalog number: 04-1073 )
- Phospho-T185/Y187-ERK2 [Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)] (Cell Signaling Technology, catalog number: 9101 )
- Phospho-S235/236-rpS6 (Cell Signaling Technology, catalog number: 2211 )
- Phospho-S209-eIF4E (Cell Signaling Technology, catalog number: 9741 )
- β-actin [AC-15] (Abcam, catalog number: AB6276 )
- Synaptoneurosome buffer (see Recipes)
Equipment
- Swinnex filter holder (Merck Millipore Corporation, catalog number: SX0002500 )
- 4 ºC Eppendorf table centrifuge
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Puighermanal, E., Biever, A. and Valjent, E. (2016). Synaptoneurosome Preparation from C57BL/6 Striata. Bio-protocol 6(4): e1735. DOI: 10.21769/BioProtoc.1735.
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Biever, A., Puighermanal, E., Nishi, A., David, A., Panciatici, C., Longueville, S., Xirodimas, D., Gangarossa, G., Meyuhas, O., Herve, D., Girault, J. A. and Valjent, E. (2015). PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons. J Neurosci 35(10): 4113-4130.
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分类
神经科学 > 细胞机理 > 突触生理学
分子生物学 > 蛋白质 > 检测
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