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Jan 2013

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Subcellular Fractionation of Mouse Brain Homogenates
小鼠脑组织匀浆的亚细胞分离   

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Abstract

This subcellular fractionation protocol is used for separation of cellular organelles based on their density. We have designed and optimized the protocol for separation of subcellular compartments of brain homogenates with focus on the localization and trafficking of transmembrane proteins, but we have also successfully used this protocol for fractionation of other types of tissue. The protocol has two major steps 1) preparation of homogenate from dissected tissue and 2) separation of organelles by centrifugation of homogenates using a continuous sucrose gradient.

Materials and Reagents

  1. Mice
  2. PBS
  3. Phosphatase inhibitor (e.g. PhosStop) (F. Hoffmann-La Roche, catalog number: 04906845001 )
  4. HEPES
  5. EDTA
  6. Distilled water
  7. Glucose
  8. Protease inhibitors (e.g. Complete) (F. Hoffmann-La Roche, catalog number: 11697498001 )
  9. Solution A (see Recipes)
  10. 2.5 M sucrose stock (see Recipes)
  11. Sucrose solution (0.8 M and 1.6 M) (see Recipes)

Equipment

  1. A pair of scissor
  2. A brain scooper
  3. Falcon tubes (15 ml)
  4. Centrifuge
  5. Ultracentrifuge
  6. Ultracentrifuge rotor (T70.1 Ti and Sw41Ti )
  7. Ultracentrifuge tubes for T70.1 Ti rotor (Polycarbonate, 16 x 76 mm) (Beckman Coulter, catalog number: 355651 )
  8. Ultracentrifuge tubes for SW41 Ti rotor (14 x 89 mm) (Seton Open-Top Polyclear centrifuge tubes, catalog number: 7030 )
  9. Thomas® Teflon Pestle (A.H Thomas Co., model: 3431-E20 )
  10. 1 and 10 ml syringes
  11. Needles (18 and 27 G)
  12. Gradient Master 107 ip (BioComp, model: 107 ) with marker block and cannula
  13. Refractometer

Procedure

  1. Mouse is sacrificed by cervical dislocation; Restrain the mouse on a hard, flat surface. Hold a strong stick or metal rod firmly against the base of the skull, and the tail firmly with the other hand. Pull the mouse body away from the head in one single quick motion. Verify the dislocation by feeling for a separation between cervical vertebras.
  2. Cut the head off the mouse, and open the skin of the head with a pair of scissor.
  3. Open the skull of the mouse using a pair of scissor, and gently lift out the exposed brain using a brain scooper and transfer to ice-cold PBS.
  4. Brains are quickly rinsed in ice-cold PBS.
  5. Brains are put in 10 ml solution A containing proteinase inhibitors and continue to point 9 when all brains have been isolated.
  6. Repeat steps 1-7 for the remaining mice.
  7. The brains are homogenized using a Thomas® Teflon Pestle.
  8. Transfer the homogenized brain tissue to a 15 ml Falcon tube.
  9. Centrifuge at 1,000 x g for 10 min at 4 °C.
  10. Transfer the supernatant (there are typically several layers of supernatant – take them all) to a new 15 ml Falcon tube.
  11. Centrifuge at 3,000 x g for 10 min at 4 °C.
  12. Transfer the supernatant to an ultracentrifugation tube.
  13. Centrifuge at 13,000 rpm for 10 min using an ultracentrifuge with a T70.1 Ti rotor.
  14. Transfer the supernatant to a new ultracentrifugation tube and centrifuge at 50,000 rpm for 45 min at 4 °C (using the same rotor (T70.1 Ti rotor) as previous step).
    Note: During this centrifugation step, one can prepare the sucrose gradient.
  15. Discard the supernatant.
  16. Dissolve pellet in 600 μl solution A containing proteinase inhibitors. Use a 1 ml syringe with an 18 G needle first, thereafter a 27 G needle to dissolve pellet.
  17. Carefully layer 500 μl of the homogenate (= dissolved pellet) on top of a continuous 0.8 to 1.6 M sucrose gradient and centrifuge at 25,000 rpm for 18 h/overnight, using a SW41 Ti swing bucket rotor.
    Notes for preparation of the gradient:
    1. The gradient is made with a Gradient Master 107 ip.
    2. Insert a tube in the marker block and mark a line on the tube at the half-full mark for short, 4 mm cap.
    3. Add 5.5 ml 0.8 M sucrose solution to the tube.
    4. Add 1.6 M sucrose beneath the 0.8 M sucrose solution using a cannula (a cannula is received together with the Gradient Master) and a 10 ml syringe until the bottom of the 0.8 M sucrose solution reaches the recently marked line.
    5. Adjust the level of the Gradient Master before use, to ensure that the plate is in level.
    6. Prepare the 10-57% linear gradient, by choosing “SW41” in the ”gradient menu” list of BioComp Gradient Master. This program will mix the gradient at 50 degrees for 10 min, followed by 1 min at 80 degrees.
  18. Next day the gradient is fractionated into 24 samples (500 μl/sample), using a 1 ml pipette. Alternatively, one can use a Piston Gradient Fractionator to collect the fractions.
  19. Store the fractions at -20 °C until Western blot analysis.

Notes

  1. Perform steps 1-9 as fast as possible.
  2. Do as many steps as possible on ice.
  3. Remember to tare the balance of the weight of the samples before performing ultracentrifugation. Use Solution A to adjust the weight.
  4. Stored fractions should be thawed on ice and vortexed before preparation of samples for Western blot analysis.

Recipes

  1. Solution A (200 ml)
    0.25 mM sucrose
    1 mM EDTA
    20 mM HEPES (pH 7.4)
    Mix 17 g sucrose with approximately 100 ml dH2O
    Add 400 μl EDTA (500 mM stock, to a final concentration of 1 mM)
    Add 8 ml HEPES (500 mM stock, to a final concentration of 20 mM)
    Add dH2O to a final volume of 200 ml
    pH 7.4
    Store at 4 °C
  2. 2.5 M sucrose stock (580 ml)
    500 g sucrose
    Add a little volume of distilled water at the time, while heating, until final volume is approximately 580 ml.
    Measure refractive index to ensure a concentration of approximately 2.5 M
    Store at 4 °C.
  3. 0.8 M sucrose solution (50 ml) in 10 mM HEPES (pH 7.2)
    16 ml of 2.5 M sucrose stock
    1 ml HEPES (500 mM stock, to a final concentration of 10 mM) (pH 7.2)
    Add 33 ml dH2O to a final volume of 50 ml
    Mix all ingredients
    Store at 4 °C if the solution is made shortly before experiment, otherwise the solution can be stored at -20 °C
  4. 1.6 M Sucrose solution (50 ml) in 10 mM HEPES (pH 7.2)
    32 ml of 2.5 M sucrose stock
    1 ml HEPES (500 mM stock, to a final concentration of 10 mM) (pH 7.2)
    Add 17 ml dH2O to a final volume of 50 ml
    Mix all ingredients
    Store at 4 °C if the solution is made shortly before experiment, otherwise the solution can be stored at -20 °C

References

  1. Gustafsen, C., Glerup, S., Pallesen, L. T., Olsen, D., Andersen, O. M., Nykjaer, A., Madsen, P. and Petersen, C. M. (2013). Sortilin and SorLA display distinct roles in processing and trafficking of amyloid precursor protein. J Neurosci 33(1): 64-71.

简介

这种亚细胞分馏方案用于根据细胞密度分离细胞器。 我们设计和优化了用于分离脑匀浆物的亚细胞区域的方案,重点是跨膜蛋白的定位和运输,但是我们也已经成功地使用该方案来分离其他类型的组织。 方案有两个主要步骤:1)从解剖组织制备匀浆物; 2)通过使用连续蔗糖梯度离心匀浆分离细胞器。

材料和试剂

  1. 小鼠
  2. PBS
  3. 磷酸酶抑制剂(例如PhosStop)(F.Hoffmann-La Roche,目录号:04906845001)
  4. HEPES
  5. EDTA
  6. 蒸馏水
  7. 葡萄糖
  8. 蛋白酶抑制剂(例如,Complete)(F.Hoffmann-La Roche,目录号:11697498001)
  9. 解决方案A(参见配方)
  10. 2.5 M蔗糖原料(见配方)
  11. 蔗糖溶液(0.8 M和1.6 M)(参见配方)

设备

  1. 一双剪刀
  2. 大脑罩
  3. Falcon管(15ml)
  4. 离心机
  5. 超速离心机
  6. 超速离心机转子(T70.1 Ti和Sw41Ti)
  7. 用于T70.1Ti转子的超速离心管(聚碳酸酯,16×76mm)(Beckman Coulter,目录号:355651)
  8. 用于SW41 Ti转子(14×89mm)(Seton Open-Top Polyclear离心管,目录号:7030)的超速离心管
  9. Thomas ®特氟龙杵(A.H Thomas Co.,型号:3431-E20)
  10. 1和10ml注射器
  11. 针(18和27 G)
  12. Gradient Master 107ip(BioComp,型号:107),带有标记块和插管
  13. 折射计

程序

  1. 通过颈脱位处死小鼠; 将鼠标放在坚硬,平坦的表面上。 用坚固的棍子或金属杆牢牢地靠在颅骨的基部,用另一只手牢固地固定尾部。 通过一次快速动作将鼠标身体从头部拉出。 通过感觉到颈椎之间的分离来验证脱臼
  2. 剪下的小鼠的头,用一把剪刀打开头部的皮肤。
  3. 使用一把剪刀打开鼠标的头骨,轻轻地使用大脑舀出暴露的大脑,并转移到冰冷的PBS。
  4. 大脑在冰冷的PBS中快速冲洗
  5. 将大脑置于10ml含有蛋白酶抑制剂的溶液A中,并在所有大脑分离后继续指向9
  6. 对剩余的小鼠重复步骤1-7。
  7. 使用Thomas Teflon Pestle将脑匀浆。
  8. 将匀浆的脑组织转移到15毫升Falcon管
  9. 在4℃下以1,000xg离心10分钟。
  10. 转移上清液(通常有几层上清液 - 把它们全部)到一个新的15毫升Falcon管
  11. 在4℃下以3,000xg离心10分钟。
  12. 将上清液转移到超速离心管中。
  13. 使用具有T70.1Ti转子的超速离心机在13,000rpm离心10分钟。
  14. 转移上清液到新的超速离心管,并在50,000 rpm离心45分钟在4°C(使用相同的转子(T70.1 Ti转子)如前一步骤)。
    注意:在这个离心步骤中,可以制备蔗糖梯度。
  15. 弃去上清液。
  16. 溶解颗粒在600μl含蛋白酶抑制剂的溶液A.使用1毫升注射器先用18 G针,然后用27 G针溶解小丸
  17. 小心地将500μl匀浆(=溶解沉淀)层叠在连续的0.8至1.6M蔗糖梯度的顶部上,并使用SW41Ti摆动叶片转子以25,000rpm离心18小时/过夜。
    准备渐变的注意事项:

    1. 在标记块中插入一个管,在管的半满标记处标记一条4毫米的短帽。
    2. 向管中加入5.5ml 0.8M蔗糖溶液。
    3. 使用套管(与Gradient Master一起接受套管)和10ml注射器,在0.8M蔗糖溶液下添加1.6M蔗糖,直到0.8M蔗糖溶液的底部达到最近标记的线。
    4. 通过在BioComp Gradient Master的"渐变菜单"列表中选择"SW41"来准备10-57%的线性梯度。 该程序将混合梯度在50度10分钟,然后在80度1分钟。
  18. 第二天,使用1ml移液管将梯度分级为24个样品(500μl/样品)。 或者,可以使用活塞梯度分馏器收集馏分。
  19. 将馏分储存在-20℃,直到蛋白质印迹分析

笔记

  1. 尽快执行步骤1-9。
  2. 在冰上尽可能多的步骤。
  3. 记得在进行超速离心之前称重样品的重量平衡。 使用溶液A调整重量。
  4. 储存的级分应在冰上解冻并涡旋,然后制备用于Western印迹分析的样品。

食谱

  1. 溶液A(200ml)
    0.25mM蔗糖 1mM EDTA
    20mM HEPES(pH7.4) 将17g蔗糖与约100ml dH 2 O混合 加入400μlEDTA(500mM储液,最终浓度为1mM) 加入8ml HEPES(500mM储备液,至终浓度为20mM) 将dH 2 O加至最终体积为200ml
    pH 7.4
    存储在4°C
  2. 2.5M蔗糖原液(580ml) 500克蔗糖
    此时加入少量蒸馏水,同时加热,直到最终体积约580 ml 测量折射率,以确保浓度约为2.5 M/m 储存于4°C。
  3. 0.8M蔗糖溶液(50ml)在10mM HEPES(pH7.2)中的溶液 16ml 2.5M蔗糖储备液
    1ml HEPES(500mM储液,最终浓度为10mM)(pH7.2) 加入33ml dH 2 O至最终体积为50ml
    混合所有成分
    如果溶液是在实验前不久在4°C储存,否则溶液可以储存在-20°C
  4. 1.6M蔗糖溶液(50ml)在10mM HEPES(pH7.2)中的溶液 32ml 2.5M蔗糖储备液
    1ml HEPES(500mM储液,最终浓度为10mM)(pH7.2) 加入17ml dH 2 O至最终体积为50ml
    混合所有成分
    如果溶液是在实验前不久在4°C储存,否则溶液可以储存在-20°C

参考文献

  1. Gustafsen,C.,Glerup,S.,Pallesen,L.T.,Olsen,D.,Andersen,O.M.,Nykjaer,A.,Madsen,P.and Petersen, Sortilin和SorLA在淀粉样前体蛋白的加工和运输中显示出不同的作用。 J Neurosci 33(1):64-71
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 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. Olsen, D. and Gustafsen, C. (2013). Subcellular Fractionation of Mouse Brain Homogenates. Bio-protocol 3(15): e839. DOI: 10.21769/BioProtoc.839.
  2. Gustafsen, C., Glerup, S., Pallesen, L. T., Olsen, D., Andersen, O. M., Nykjaer, A., Madsen, P. and Petersen, C. M. (2013). Sortilin and SorLA display distinct roles in processing and trafficking of amyloid precursor protein. J Neurosci 33(1): 64-71.
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