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Microsome Isolation from Tissue
从组织中分离微粒体   

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Abstract

This protocol details the extraction of microsomes from frozen tissue in order to further examine the protein-protein interactions occurring within the endoplasmic reticulum. This protocol was adapted from Abisambra et al. (2013) with modifications made in order to optimize for subsequent use.

Keywords: Endoplasmic reticulum(内质网), Microsomes(微粒体), Sub-cellular fractionation(亚细胞分馏)

Materials and Reagents

  1. Sucrose
  2. Protease Inhibitor cocktail, EDTA free (Merck KGaA, Calbiochem, catalog number: 539134 )
  3. Phosphatase inhibitor cocktail II
  4. Phosphatase inhibitor cocktail III
  5. PMSF at 10 mM in DMSO or 1.74 mg/ml (Thermo Fisher Scientific, catalog number: 36978 )
  6. Phosphatase Arrest II cocktail (Geno Technology, catalog number: 786-451 )
  7. Phosphatase Arrest III cocktail (Geno Technology, catalog number: 786-452 )
  8. M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, catalog number: 78501 )

Equipment

  1. Sterile bottle filter
  2. Glass Dounce homogenizer
  3. Refrigerated centrifuge
  4. Microfuge tubes rated for at least 25,000 x g centrifugation

Procedure

  1. Make a 0.25 M sucrose solution that contains protease inhibitor cocktail, phosphatase inhibitor cocktails II and III, and PMSF as follows:
    Per 100 μl of Sucrose master mix add:
    a.  96 μl of 0.25 M sucrose
    b.  1 μl of protease inhibitor cocktail
    c.  1 μl of phosphatase inhibitor cocktail II
    d.  1 μl of phosphatase inhibitor cocktail III
    e.  1 μl of PMSF
  2. Weigh tissue to be analyzed and add 10x its mass in volume of sucrose master mix (see step 1; i.e. 100 mg = 1,000 μl of sucrose solution).
  3. While keeping all solutions on ice, add the appropriate amount of sucrose solution to tissue and dounce homogenize until a completely homogenous solution is obtained.
  4. Spin the homogenate at 10,000 x g for 10 min at 4 °C.
  5. Transfer the supernatants to a new microfuge tube (save the pellet at -20 °C) and spin at 30,000 x g for 90 min in a fixed angle rotor (or at 25,800 x g for 2 h).
  6. Transfer the supernatant to a different microfuge tube and save at -20 °C. The remaining pellet corresponds to the microsomal fraction.
  7. Pipette gently to resuspend the microsome pellet in 200 μl of the following mix (per 100 μl):
    a. 96 μl of MPER buffer
    b. 1 μl of protease inhibitor cocktail
    c. 1 μl of phosphatase arrest cocktail II
    d. 1 μl of phosphatase arrest cocktail III
    e. 1 μl of PMSF

Acknowledgments

We thank Dr. Gene Ness, Dr. Huntington Potter, and Dr. Chad Dickey for supporting the development and adaptation of this protocol in their labs. We credit the following article for this work: Abisambra et al. (2013). Financial support during the time of protocol development came from the Alzheimer’s Association NIRGD-12-242642, the Foundation for PSP/CBD and Related Brain Disorders (6144107400), and NIH/NIA ADC Pilot Grant from 5P30AG028383-08.

References

  1. Abisambra, J. F., Jinwal, U. K., Blair, L. J., O'Leary, J. C., 3rd, Li, Q., Brady, S., Wang, L., Guidi, C. E., Zhang, B., Nordhues, B. A., Cockman, M., Suntharalingham, A., Li, P., Jin, Y., Atkins, C. A. and Dickey, C. A. (2013). Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation. J Neurosci 33(22): 9498-9507.
  2. Abisambra, J. F., Fiorelli, T., Padmanabhan, J., Neame, P., Wefes, I. and Potter, H. (2010). LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease. PLoS One 5(1): e8556.

简介

该协议详细描述了从冷冻组织中提取微粒体,以进一步检查在内质网内发生的蛋白质 - 蛋白质相互作用。 该方案由Abisambra等人 (2013)进行了修改,以优化后续使用。

关键字:内质网, 微粒体, 亚细胞分馏

材料和试剂

  1. 蔗糖
  2. 蛋白酶抑制剂混合物,无EDTA(Merck KGaA,Calbiochem,目录号:539134)
  3. 磷酸酶抑制剂鸡尾酒II
  4. 磷酸酶抑制剂鸡尾酒III
  5. 在DMSO中为10mM的PMSF或1.74mg/ml(Thermo Fisher Scientific,目录号:36978)
  6. 磷酸酶Arrest II鸡尾酒(Geno Technology,目录号:786-451)
  7. 磷酸酶Arrest III混合物(Geno Technology,目录号:786-452)
  8. M-PER哺乳动物蛋白提取试剂(Thermo Fisher Scientific,目录号:78501)

设备

  1. 无菌瓶过滤器
  2. 玻璃Dounce匀浆器
  3. 冷冻离心机
  4. 定量至少25,000×g离心的微量离心管

程序

  1. 制备含有蛋白酶抑制剂混合物,磷酸酶抑制剂鸡尾酒II和III和PMSF的0.25 M蔗糖溶液,如下:
    每100微升蔗糖主混合物添加:
    一个。  96μl0.25M蔗糖
    b。   1μl蛋白酶抑制剂混合物
    C。   1μl磷酸酶抑制剂鸡尾酒II
    d。   1μl磷酸酶抑制剂鸡尾酒III
    e。   1μl的PMSF
  2. 称量要分析的组织,并加入其体积为蔗糖主混合物体积的10倍(参见步骤1;即,100mg =1,000μl蔗糖溶液)。
  3. 在将所有溶液保持在冰上的同时,向组织中加入适量的蔗糖溶液,并且使其均匀化,直到获得完全均匀的溶液。
  4. 在4℃下以10,000×g离心匀浆10分钟
  5. 将上清液转移到新的微量离心管中(在-20℃下保存沉淀),并在固定角度的转子中以30,000×g旋转90分钟(或在25,800×g < 2小时)。
  6. 转移上清液到不同的微量离心管,在-20°C保存。 剩余的颗粒对应于微粒体部分
  7. 轻轻移液以将微粒体沉淀物重悬于200μl以下混合物(每100μl)中:
    一个。 96μlMPER缓冲液
    b。 1μl蛋白酶抑制剂混合物
    C。 1微升磷酸酶停止鸡尾酒II
    d。 1微升磷酸酶停止鸡尾酒III
    e。 1μlPMSF

致谢

我们感谢Gene Ness博士,Huntington Potter博士和Chad Dickey博士在实验室中支持本协议的开发和适应。 我们认为这项工作的下列文章:Abisambra 等人(2013)。 方案开发期间的资金支持来自阿尔茨海默氏病协会NIRGD-12-242642,PSP/CBD和相关脑病症基金会(6144107400)和来自5P30AG028383-08的NIH/NIA ADC试验授予。

参考文献

  1. 本研究结果表明,该方法能够有效地提高患者的生活质量,提高患者的生活质量, M.,Suntharalingham,A.,Li,P.,Jin,Y.,Atkins,CA和Dickey,CA(2013)。 Tau积累通过损害内质网相关的降解来激活展开的蛋白反应。 J Neurosci 33(22):9498-9507。
  2. Abisambra,J.F.,Fiorelli,T.,Padmanabhan,J.,Neame,P.,Wefes,I。和Potter,H。(2010)。 LDLR表达和定位在阿尔茨海默病的小鼠和人类细胞培养模型中有所改变。 PLoS One 5(1):e8556。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bodero, M. and Abisambra, J. F. (2014). Microsome Isolation from Tissue. Bio-protocol 4(3): e1038. DOI: 10.21769/BioProtoc.1038.
  2. Abisambra, J. F., Jinwal, U. K., Blair, L. J., O'Leary, J. C., 3rd, Li, Q., Brady, S., Wang, L., Guidi, C. E., Zhang, B., Nordhues, B. A., Cockman, M., Suntharalingham, A., Li, P., Jin, Y., Atkins, C. A. and Dickey, C. A. (2013). Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation. J Neurosci 33(22): 9498-9507.
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