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Preparation of Pre- and Post-synaptic Density Fraction from Mouse Cortex
从小鼠皮质中制备突触前和突出后的致密区   

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Abstract

The understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. Here we described an efficient way to isolate the crude synaptosome, presynaptic fraction, and PSD fraction. It helps to identify the location of synaptic protein and find the potential synaptic complex.

Materials and Reagents

  1. Sucrose
  2. Protease inhibitor cocktail (1:100) (Sigma-Aldrich, catalog number: P8340-5ml )
  3. 20 mM HEPES (pH 7.0)
  4. Triton X-100
  5. KCl
  6. NaHCO3
  7. MgCl2
  8. CaCl2
  9. Tris-HCl
  10. SDS
  11. Glycerol
  12. 2-mercaptoethanol
  13. BPB
  14. Solution A (see Recipes)
  15. SDS-PAGE sample buffer (see Recipes)

Equipment

  1. Eppendorf table centrifuge
  2. Swinging bucket rotor (model: SW51Ti )
  3. Fixed-angle rotor
  4. Dounce mini-homogenizer

Procedure

          Note: PSD fraction of mouse cortex was prepared according to modified protocol.

  1. Corticles are taken in the cold PBS under microscope. Could store at -80 °C if not using immediately. Brain is homogenized in Dounce mini-homogenizer, 50 strokes. Dissect 1 half cortex (or 2 Hipp), add 4 ml buffer to homogenize to looks milky and no obvious pieces of tissue.
    If not indicated below, all experiments are performed at 4 °C.
  2. The homogenates were centrifuged at 470 x g for 2 min.
  3. Resultant supernatants (S1 fraction) were centrifuged at 10,000 x g for 10 min to obtain mitochondria- and synaptosome-enriched pellets (P2) and supernatants (S2 fraction) containing soluble proteins.
  4. P2 fractions were resuspended in 3.75 ml of 0.32 M sucrose, which was then layered onto 0.8 M sucrose. Centrifuge at 9,100 x g for 15 min in a swinging bucket rotor.
  5. After centrifugation, synaptosomes (most of the loose pellets) were collected from 0.8 M sucrose layer (Figure 1) and resuspended with equal volume of 20 mM HEPES (pH 7.0), 2% Triton X-100 and 150 mM KCl.


    Figure 1. Sucrose ultracentrifugation of PSD fraction.

  6. Samples were centrifuged at 20,800 x g for 45 min using a fixed-angle rotor, and resulting supernatants were collected as presynaptic fraction.
  7. Pellets were resuspended in a solution of 1% Triton X-100 and 75 mM KCl using a Dounce mini-homogenizer and centrifuged again at 20,800 x g for 30 min to yield final pellets (PSD fraction, which be identified by marker PSD95), which were washed with 20 mM HEPES and dissolved in 1x SDS-PAGE sample buffer.

Recipes

  1. Solution A
    0.32 M sucrose
    1 mM NaHCO3
    1 mM MgCl2
    0.5 mM CaCl2
    1 mM PMSF and protease inhibitors
  2. SDS-PAGE sample buffer
    0.125 M Tris-HCl (pH 6.8)
    4% SDS
    20% Glycerol
    10% 2-mercaptoethanol
    0.2% BPB

Acknowledgments

This work was supported in part by grants from American Heart Association (C.Y). Methods are previously simply described in Tao et al. (2013).

References

  1. Tao, Y., Chen, Y. J., Shen, C., Luo, Z., Bates, C. R., Lee, D., Marchetto, S., Gao, T. M., Borg, J. P., Xiong, W. C. and Mei, L. (2013). Erbin interacts with TARP gamma-2 for surface expression of AMPA receptors in cortical interneurons. Nat Neurosci 16(3): 290-299.

简介

突触后信号系统在兴奋性突触的组织的理解已通过在突触后密度(PSD)部分,富含结构与PSD的形态的亚细胞部分中的蛋白质的识别辅助。 在这里我们描述了一种有效的方式来隔离粗糙的突触体,突触前分数和PSD分数。 它有助于确定突触蛋白的位置,并找到潜在的突触复杂

材料和试剂

  1. 蔗糖
  2. 蛋白酶抑制剂混合物(1:100)(Sigma-Aldrich,目录号:P8340-5ml)
  3. 20mM HEPES(pH 7.0)
  4. Triton X-100
  5. KCl
  6. NaHCO 3
  7. MgCl 2
  8. CaCl <2>
  9. Tris-HCl
  10. SDS
  11. 甘油
  12. 2-巯基乙醇
  13. BPB
  14. 解决方案A(参见配方)
  15. SDS-PAGE样品缓冲液(参见配方)

设备

  1. Eppendorf台式离心机
  2. 摆动转子(型号:SW51Ti)
  3. 定角转子
  4. Dounce微型均化器

程序

          注意:根据修改的方案制备小鼠皮质的PSD级分。

  1. 在显微镜下在冷PBS中取出皮质。 如果不立即使用,可储存在-80°C。 将脑在Dounce微型匀浆器中匀浆50次。 解剖1半皮质(或2 Hipp),加4毫升缓冲液 均匀化为看起来乳白色,没有明显的组织碎片。
    如果以下没有指出,所有实验在4℃下进行。
  2. 匀浆在470×g离心2分钟。
  3. 将所得上清液(S1级分)以10,000×g离心10分钟,以获得富含线粒体和突触小体的沉淀(P2)和含有可溶性蛋白质的上清液(S2级分)。
  4. 将P2级分重悬浮于3.75ml 0.32M蔗糖中,然后将其分层到0.8M蔗糖上。在摇摆转子中以9,100×g离心15分钟。
  5. 离心后,从0.8M蔗糖层(图1)收集突触体(大多数松散的沉淀),并用等体积的20mM HEPES(pH 7.0),2%Triton X-100和150mM KCl重悬。


    图1. PSD级分的蔗糖超速离心。

  6. 使用固定角转子将样品在20,800×g离心45分钟,收集所得上清液作为突触前级分。
  7. 使用Dounce微型匀浆器将沉淀重悬浮于1%Triton X-100和75mM KCl的溶液中,并再次在20,800×g离心30分钟,得到最终沉淀(PSD级分,其被鉴定 通过标记PSD95),其用20mM HEPES洗涤并溶解在1x SDS-PAGE样品缓冲液中

食谱

  1. 解决方案A
    0.32 M蔗糖 1mM NaHCO 3/v/v 1mM MgCl 2
    0.5mM CaCl 2·h/v 1mM PMSF和蛋白酶抑制剂
  2. SDS-PAGE样品缓冲液
    0.125M Tris-HCl(pH 6.8)
    4%SDS
    20%甘油
    10%2-巯基乙醇 0.2%BPB

致谢

这项工作部分得到了美国心脏协会(C.Y)的资助。 方法之前简单描述在Tao等人(2013)中。

参考文献

  1. Tao,Y.,Chen,YJ,Shen,C.,Luo,Z.,Bates,CR,Lee,D.,Marchetto,S.,Gao,TM,Borg,JP,Xiong,WC和Mei, 2013)。 Erbin与TARP gamma-2相互作用,用于皮质中间神经元中AMPA受体的表面表达。 Nat Neurosci 16(3):290-299。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shen, C. and Chen, Y. (2013). Preparation of Pre- and Post-synaptic Density Fraction from Mouse Cortex. Bio-protocol 3(17): e880. DOI: 10.21769/BioProtoc.880.
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