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Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords
小鼠脑和脊髓中可溶性和不溶性蛋白质组分的提取   

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Abstract

The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western blotting.

Keywords: Protein extraction(蛋白质提取), Homogenization(匀化), Soluble fraction(可溶性组分), SDS(SDS), Brain(脑)

Background

This simple and reproducible protocol of brain tissue protein fractionation details the initial separation of a total protein homogenate into a soluble and an insoluble fraction. It can also be applied also to other tissue samples and yields a soluble fraction containing hydrophilic proteins and an insoluble fraction consisting of more hydrophobic proteins. Following an initial homogenization in a lysis buffer containing no detergent, the supernatant including the soluble protein fraction is removed and the pellet containing the insoluble fraction can be further extracted using Sodium Dodecyl Sulfate (SDS) as detergent to ensure entire cell lysis (see Figure 1). This approach can facilitate the analysis of low-abundance proteins by reducing the complexity of the sample.


Figure 1. Flow-chart describing the sequential extraction procedure

Materials and Reagents

  1. Pipette tips
    10 µl pipette tips (SARSTEDT, catalog number: 70.1130 )
    200 µl pipette tips (SARSTEDT, catalog number: 70.760.002 )
    1,000 µl pipette tips (SARSTEDT, catalog number: 70.762 )
  2. Reaction tubes
    1.5 ml tubes (SARSTEDT, catalog number: 72.706 )
    2 ml tubes (SARSTEDT, catalog number: 72.691 )
  3. Mice (protocol has been tested with male and female C57Bl/6 mice of 8-52 weeks)
  4. Sodium chloride (NaCl) (≥ 99.5%) (Carl Roth, catalog number: 3957 )
  5. Tris (≥ 99.9%, p.a.) (Carl Roth, catalog number: 4855 )
  6. Protease inhibitor cocktail (cOmpleteTM Mini EDTA-free EasyPack) (Roche Diagnostics, catalog number: 04693159001 )
  7. Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
  8. Sodium dodecyl sulfate (SDS) (≥ 99.5%) (Carl Roth, catalog number: 2326 )
  9. Benzonase (Sigma-Aldrich, catalog number: E1014 )
  10. Lysis buffer (see Recipes)
  11. 2% SDS (see Recipes)

Equipment

  1. Pipettes (Research 0.5-10 µl, 10-100 µl, 100-1,000 µl) (Eppendorf)
  2. Glass homogenizer (2 ml) (Carl Roth, catalog number: TT57.1 )
  3. Teflon pestle (Carl Roth, catalog number: TT63.1 )
  4. Stirring device (Ingenieurbüro CAT M. Zipperer, model: R 50D )
  5. Heraeus Biofuge Stratos ( Rotor) (Heraeus Holding, catalog number: 3332 )
  6. Ultrasound sonicator (Emerson Electric, BRANSON, model: 150 )

Procedure

Notes:

  1. The following protocol has been used to extract soluble and insoluble protein fractions from mouse brain and spinal cord samples from mouse models of Alzheimer’s disease (see Härtig et al. [2014] and Saul and Wirths [2017]). The user might need to modify the amount of solutions (e.g., lysis buffer) depending on the particular downstream application (e.g., Western blotting or ELISA). For Western blots, we recommend using 20 to 50 µg of total protein per well. This procedure will yield soluble protein fractions with concentrations of approx. 2-4 µg/µl and SDS-soluble fractions of approx. 5-8 µg/µl. Depending on the initial tissue weight, protein amounts > 2 mg (soluble fraction) and > 4 mg (SDS-soluble fraction) will be usually achieved.
  2. For mouse brain and/or spinal cord preparation, animals were anesthetized by CO2 and sacrificed by cervical dislocation. Tissues were carefully dissected and either directly used or kept at -80 °C until further use.
  1. Tissue homogenization
    1. Mechanically homogenize fresh or deep-frozen brain or spinal cord tissue in a weight:volume ratio of 1:7 using cold lysis buffer (700 µl buffer per 100 mg tissue, see Recipes) with 10 strokes in glass-Teflon homogenizer (Figure 2) at 800 rpm (Figures 3A and 3B). The homogenate should have a milky appearance without visible particles.
      Note: Our lab uses one brain hemisphere with cerebellum and olfactory bulb removed and flash frozen on a plate of dry ice. We do not remove the meninges upon harvesting the tissue.


      Figure 2. Motor-driven homogenizer


      Figure 3. Exemplary images illustrating the different homogenization and centrifugation steps

  2. Protein extraction
    1. Transfer the entire homogenate to 1.5 ml reaction tubes (Figure 3C).
    2. Spin the samples at 17,000 x g for 20 min at 4 °C.
      Note: Centrifuge should be pre-chilled to 4 °C.
    3. Remove the supernatant (soluble fraction) and store at -80 °C until further use (Figure 3D).
    4. Resuspend pellet 1 (Figure 3E) in either 800 µl (brain hemisphere) or 200 µl (spinal cord) 2% SDS (see Recipes) with a few short ultrasound pulses via sonication on ice. The pellet should dissolve upon this treatment (Figure 3F).
      Note: We use 5 short ultrasound pulses of approx. 1 sec length at level 2 in 1.5 ml reaction tubes. The clear SDS solution will turn turbid. Be careful to prevent foam formation.
    5. Add 1 µl of Benzonase, gently mix and incubate at room temperature for 5 min to remove DNA.
    6. Spin the samples at 17,000 x g for 20 min at 4 °C.
    7. Transfer the supernatant (insoluble fraction) to a fresh 1.5 ml reaction tube and store at -80 °C until further use (Figure 3G).
    8. Pellet 2 could be stored at -80 °C for further extraction using e.g., formic acid.

Data analysis

Protein fractions obtained by this protocol can be further used in downstream applications like Western blot or enzyme-linked immunosorbent assays (ELISA) (for an example of potential further use in Western blots see Härtig et al., 2014; Saul and Wirths, 2017).

Recipes

  1. Lysis buffer
    120 mM NaCl
    50 mM Tris
    cOmpleteTM protease inhibitor cocktail (1 tablet per 10 ml)
    Phosphatase inhibitor cocktail 3 (use at 1:100 dilution)
    Adjust to pH 8.0
    Note: Can be stored at 4 °C for 1 month or at -20 °C for up to 3 months.
  2. 2% SDS
    Dissolve 1 g of SDS in 10 ml of ddH2O and prepare a 1:5 dilution
    Note: SDS consists of fine crystals that easily disperse. Wear a mask when weighing SDS and clean equipment thoroughly after use.

Acknowledgments

Financial support from the Alzheimer Forschung Initiative e.V. is gratefully acknowledged.

References

  1. Härtig, W., Saul, A., Kacza, J., Grosche, J., Goldhammer, S., Michalski, D. and Wirths, O. (2014). Immunolesion-induced loss of cholinergic projection neurones promotes β-amyloidosis and tau hyperphosphorylation in the hippocampus of triple-transgenic mice. Neuropathol Appl Neurobiol 40(2): 106-120.
  2. Saul, A. and Wirths, O. (2017). Endogenous apolipoprotein E (ApoE) fragmentation is linked to amyloid pathology in transgenic mouse models of Alzheimer's disease. Mol Neurobiol 54(1): 319-327.

简介

目前的方案详述了从小鼠脑脊髓样品中制备可溶性和不溶性蛋白质裂解物。 详细地描述了组织匀浆和顺序蛋白质提取。 该方法产生可溶性和不溶性蛋白质提取物,其可以在下游应用中进一步加工,如ELISA或Western印迹法。
【背景】这种简单且可重现的脑组织蛋白分离方案详述了总蛋白匀浆物初始分离成可溶性和不溶性级分。 它也可以应用于其他组织样品,并产生含有亲水性蛋白质的可溶性级分和由更疏水的蛋白质组成的不溶性级分。 在不含洗涤剂的裂解缓冲液中进行初始均化后,除去含有可溶性蛋白质级分的上清液,并且可以使用十二烷基硫酸钠(SDS)作为洗涤剂进一步提取含有不溶性级分的沉淀物,以确保全细胞裂解(参见图1)。 这种方法可以通过降低样品的复杂性来促进低丰度蛋白质的分析。


图1.描述顺序提取过程的流程图

关键字:蛋白质提取, 匀化, 可溶性组分, SDS, 脑

材料和试剂

  1. 移液器提示
    10μl移液器吸头(SARSTEDT,目录号:70.1130)
    200μl移液器吸头(SARSTEDT,目录号:70.760.002)
    1,000μl移液器吸头(SARSTEDT,目录号:70.762)
  2. 反应管
    1.5ml管(SARSTEDT,目录号:72.706)
    2毫升管(SARSTEDT,目录号:72.691)
  3. 小鼠(方案已经测试了雄性和雌性C57Bl / 6小鼠8-52周)
  4. 氯化钠(NaCl)(≥99.5%)(Carl Roth,目录号:3957)
  5. Tris(≥99.9%,p.a.)(Carl Roth,目录号:4855)
  6. 蛋白酶抑制剂混合物(cOmplete TM 不含EDTA的EasyPack)(Roche Diagnostics,目录号:04693159001)
  7. 磷酸酶抑制剂混合物3(Sigma-Aldrich,目录号:P0044)
  8. 十二烷基硫酸钠(SDS)(≥99.5%)(Carl Roth,目录号:2326)
  9. Benzonase(Sigma-Aldrich,目录号:E1014)
  10. 裂解缓冲液(见配方)
  11. 2%SDS(参见食谱)

设备

  1. 移液器(研究0.5-10μl,10-100μl,100-1,000μl)(Eppendorf)
  2. 玻璃均化器(2ml)(Carl Roth,目录号:TT57.1)
  3. 铁氟龙杵(Carl Roth,目录号:TT63.1)
  4. 搅拌装置(IngenieurbüroCAT M.Ziperer,型号:R 50D)
  5. Heraeus Biofuge Stratos(转子)(Heraeus Holding,目录号:3332)
  6. 超声波超声波发生器(艾默生电气,BRANSON,型号:150)

程序

注意:

  1. 已经使用以下方案从阿尔茨海默病小鼠模型的小鼠脑和脊髓样品中提取可溶性和不溶性蛋白质级分(参见Härtig等人[2014]和Saul and Wirths [2017])。用户可能需要根据具体的下游应用(例如,Western印迹或ELISA)来修饰溶液的量(例如裂解缓冲液)。对于蛋白质印迹,我们建议每孔使用20至50μg的总蛋白。该方法将产生可溶性蛋白质级分,浓度约为2-4μg/μl和SDS-可溶级分5-8μg/μl。根据初始组织重量,蛋白质量> 2mg(可溶性级分)和>通常可以实现4mg(SDS-可溶性级分)。
  2. 对于小鼠脑和/或脊髓制备,通过CO 2 2将动物麻醉,并通过颈脱位处死。小心切开组织,直接使用或保存在-80°C直至进一步使用。
  1. 组织匀浆
    1. 使用冷裂解缓冲液(每100 mg组织700μl缓冲液,参见食谱),在玻璃 - 特氟隆匀浆器中用10次冲洗,以重量:体积比为1:7,以机械方式匀浆新鲜或深冻的脑或脊髓组织(图2)在800rpm(图3A和3B)。匀浆应具有乳白色外观,无可见颗粒。
      注意:我们的实验室使用一个脑半球,小脑和嗅球去除,并在一块干冰上快速冷冻。我们在收集组织时不要去除脑膜。


      图2.电动均质器


      图3.示出不同匀化和离心步骤的示例图像

  2. 蛋白质提取
    1. 将整个匀浆转移到1.5ml反应管中(图3C)。
    2. 在4℃下将样品以17,000 x g 旋转20分钟。
      注意:离心机应预冷至4°C。
    3. 去除上清液(可溶性级分),并储存在-80°C直到进一步使用(图3D)
    4. 通过在冰上超声处理,用几个短的超声脉冲将颗粒1(图3E)重悬于800μl(脑半球)或200μl(脊髓)2%SDS中(参见食谱)。这种处理时颗粒应溶解(图3F) 注意:我们使用约5个短的超声波脉冲。在1.5ml反应管中,在2级的1秒长度。透明的SDS溶液会变浊。小心防止泡沫形成。
    5. 加入1μlBenzonase,轻轻混匀,室温孵育5 min,去除DNA
    6. 在4℃下将样品以17,000 x g 旋转20分钟。
    7. 将上清(不溶性级分)转移到新鲜的1.5ml反应管中,并储存在-80℃直至进一步使用(图3G)。
    8. 颗粒2可以储存在-80℃以进一步提取,例如使用甲酸。

数据分析

通过该方案获得的蛋白质级分可以进一步用于下游应用如蛋白质印迹或酶联免疫吸附测定(ELISA)(例如,在Western印迹中可能进一步使用的一个例子见Härtig等人,2014年;扫罗和温斯,2017年)。

食谱

  1. 裂解缓冲液
    120 mM NaCl
    50 mM Tris
    cOmplete TM 蛋白酶抑制剂混合物(1片每10ml)
    磷酸酶抑制剂混合物3(以1:100稀释使用)
    调整至pH 8.0
    注意:可以在4°C储存1个月,或在-20°C储存3个月。
  2. 2%SDS
    将1g SDS溶解于10ml ddH 2 O中,并制备1:5稀释度 注意:SDS由易分散的细晶体组成。称重SDS后戴上面具,彻底清洁设备。

致谢

阿尔茨海默氏福祉计划e.V.的财政支持非常感谢。

参考

  1. Härtig,W.,Saul,A.,Kacza,J.,Grosche,J.,Goldhammer,S.,Michalski,D.and Wirths,O。(2014)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/23566195”target =“_ blank”>免疫激素诱导的胆碱能投射神经元的损失促进三重转基因小鼠海马中的β-淀粉样变性和tau高磷酸化。 Neuropathol Appl Neurobiol 40(2):106-120。
  2. Saul,A.和Wirths,O.(2017)。  内源性载脂蛋白E(ApoE)断裂与阿尔茨海默病转基因小鼠模型中的淀粉样蛋白病理学有关。 Mol Neurobiol 54(1):319-327。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wirths, O. (2017). Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords. Bio-protocol 7(15): e2422. DOI: 10.21769/BioProtoc.2422.
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