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Protein Flotation Assay to Isolate Lipids Rafts from Soft Tissue or Cells
蛋白质浮选分离试验从软组织或细胞中分离脂筏   

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Abstract

The arrangement in eukaryotic cell membranes of liquid-ordered states surrounded by liquid-disordered phases allows for the existence of organized membrane microdomains called rafts. Rafts play a crucial role in signal-transduction events, in lipid transport and in various internalization processes, and for all these reasons need to be purified for further characterization.

Material and Reagents

  1. Optiprep® (Optiprep® is a 60% solution of iodixanol in water, density = 1.32 g/ml) (Sigma-Aldrich, catalog number: 079K1726 )
  2. Sodium chloride (NaCl)
  3. Tris hydrochloride (Tris-HCl)
  4. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: ED255 )
  5. Triton X-100 (Sigma-Aldrich, catalog number: T-8532 )
  6. Methanol/chloroform
  7. Lysis buffer (see Recipes)
  8. Gradient buffer (see Recipes)

Equipment

  1. Pellet pestles (Sigma-Aldrich, catalog number: Z359963-1EA )
  2. Sorvall® WX90 ultracentrifuge (Thermo Fisher Scientific, catalog number: 46901 )
  3. Sorvall® TH-641 swinging bucket rotor (Thermo Fisher Scientific, catalog number: 54295 )
  4. Ultracentrifuge tubes: Beckman Coulter® open-top Ultra-ClearTM tubes (dimension: 14 x 89 mm, nominal volume: 12 ml) (Beckman Coulter®, catalog number: 344059 )
  5. 1 ml pipette
  6. Western Blotting apparatus

Procedure

  1. Extract appropriate tissue homogenates for 2 h on ice in cold lysis buffer (total protein: 1 mg in 1 ml). Soft tissue (or cell pellet) is hand-disrupted in microcentrifuge tubes with a pestle. Pestle end mates well with 1.5 ml microtubes and shaft allows gentle manual back-and-forth rotation.
  2. Assemble the gradient
    1. Prepare all solutions extemporaneously and kept on ice.
      1. Prepare a 30% Optiprep® solution in gradient buffer (1 volume of Optiprep® in 1 volume gradient buffer).
      2. Prepare a 5% Optiprep® solution in gradient buffer (1 volume of Optiprep® in 11 volumes gradient buffer).
      3. Mix the extracts (1 ml in lysis buffer) with two volumes (2 ml) of cold 60% Optiprep® to reach a final concentration of 40%.
    2. Load the extracts (3 ml in 40% Optiprep®) at the bottom of the ultracentrifuge tubes, add very slowly 6 ml of 30% Optiprep® onto the lysate and then add very slowly 2.5 ml of 5% Optiprep® onto the 30% Optiprep®.
      Note: It is important to avoid mixing of the interfaces. A good way is to let the pipet tip barely touch the surface and slowly overlay the solution while the tip is brought up along the side of the tube.
  3. Make sure the tubes (including buckets and caps) are equilibrated. Put the tubes in the buckets of the Sorvall TH-641 rotor pre-cooled at 4 °C, and screw the caps on the buckets. Spin at 100,000 x g for 24 h at 4 °C.
  4. Analyze the gradient
    1. Collect the fractions (1 ml) by gentle and slow aspiration from the meniscus at the top of the tube with an automatic 1 ml pipette, making sure that the tip of the pipette follows the lowering meniscus. Rafts should be present within the low-density fractions 2 to 4 which is the interface between the 30 and the 5% density.
    2. The equivalent of 300 μl is precipitated by the methanol/chloroform method and processed for Western Blotting. The fractions can be stored at -80 °C for up to 6 months. The protocol used for precipitation is that described in Aboulaich (2011).
    3. Always check on the distribution of raft markers in the gradient to confirm that the centrifugation has achieved a satisfactory resolution and recovery of rafts, Flotillin-1 which has been shown to be enriched in lipid rafts is the most widely used. For brain tissue, we have used prion protein which is also present in lipid rafts.

Recipes

  1. Lysis buffer
    150 mM NaCl
    25 mM Tris–HCl (pH 7.4)
    5 mM EDTA
    1% Triton X-100
    Filter sterilize and keep at 4 °C.
  2. Gradient buffer
    150 mM NaCl
    25 mM Tris–HCl (pH 7.4)
    5 mM EDTA
    Filter sterilize and keep at 4 °C.

Acknowledgments

This protocol is adapted from Lemaire-Vieille et al. (2013).

References

  1. Aboulaich, N. (2011). Protein precipitation from detergent-containing samples. Bio-protocol 1(5): e40. 
  2. Lemaire-Vieille, C., Bailly, Y., Erlich, P., Loeuillet, C., Brocard, J., Haeberle, A. M., Bombarde, G., Rak, C., Demais, V., Dumestre-Perard, C., Gagnon, J. and Cesbron, J. Y. (2013). Ataxia with cerebellar lesions in mice expressing chimeric PrP-Dpl protein. J Neurosci 33(4): 1391-1399.
  3. Persaud-Sawin, D. A., Lightcap, S. and Harry, G. J. (2009). Isolation of rafts from mouse brain tissue by a detergent-free method. J Lipid Res 50(4): 759-767.

简介

在由液体无序相包围的液体有序状态的真核细胞膜中的排列允许存在称为筏的有组织的膜微结构域。 木筏在信号转导事件,脂质运输和各种内化过程中发挥关键作用,并且由于所有这些原因,需要进行纯化以进一步表征。

材料和试剂

  1. Optiprep(Optiprep)是一种60%的碘克沙醇在水中的溶液,密度= 1.32g/ml)(Sigma-Aldrich,目录号:079K1726)
  2. 氯化钠(NaCl)
  3. Tris盐酸(Tris-HCl)
  4. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:ED255)
  5. Triton X-100(Sigma-Aldrich,目录号:T-8532)
  6. 甲醇/氯仿
  7. 裂解缓冲液(见配方)
  8. 渐变缓冲区(参见配方)

设备

  1. 丸粒杵(Sigma-Aldrich,目录号:Z359963-1EA)
  2. Sorvall WX90超速离心机(Thermo Fisher Scientific,目录号:46901)
  3. Sorvall TH-641旋转叶片转子(Thermo Fisher Scientific,目录号:54295)
  4. 超速离心管:Beckman Coulter 开顶Ultra-ClearTM管(尺寸:14×89mm,标称体积:12ml)(Beckman Coulter ,目录号:344059 )
  5. 1 ml移液器
  6. Western印迹装置

程序

  1. 提取适当的组织匀浆在冰上的冷裂解缓冲液(总蛋白:1毫克在1毫升)2小时。 用杵在微量离心管中手动破碎软组织(或细胞沉淀)。 杵末端与1.5毫升微管良好配合,轴允许温和的手动来回旋转。
  2. 组合渐变
    1. 立即准备所有溶液,并保持在冰上。
      1. 在梯度缓冲液(1体积的Optiprep在1体积梯度缓冲液中)中制备30%Optiprep溶液。
      2. 在梯度缓冲液(1体积的Optiprep在11体积梯度缓冲液中)中制备5%Optiprep溶液。
      3. 将提取物(1ml在裂解缓冲液中)与两个体积(2ml)的冷的60%Optiprep 混合,以达到40%的终浓度。
    2. 在超速离心管的底部装载提取物(3ml,在40%Optiprep中),向裂解物中非常缓慢地加入6ml 30%Optiprep,然后加入非常缓慢地将2.5ml 5%Optiprep注射到30%Optiprep上。
      注意:重要的是避免混合接口。一个好的方法是让移液管吸头几乎不接触表面,并缓慢地覆盖溶液,同时沿管的侧面提起吸头。
  3. 确保管(包括桶和盖)已平衡。将管子放在Sorvall TH-641转子的桶中,预冷却在4°C,并将盖子拧在桶上。在4℃下以100,000xg离心24小时。
  4. 分析渐变
    1. 通过使用自动1ml移液管从管的顶部缓慢和缓慢吸出来收集级分(1ml),确保移液管的尖端跟随降低的半月板。筏应存在于低密度部分2至4中,这是30和5%密度之间的界面。
    2. 通过甲醇/氯仿方法沉淀300μl的等同物,并处理用于Western印迹。 所述级分可以在-80℃下储存长达6个月。 用于降水的协议描述在Aboulaich(2011) 中。 br />
    3. 总是检查在梯度中的筏标记的分布,以确认离心已经实现令人满意的分辨率和筏的恢复,已显示富含脂筏的Flotillin-1是最广泛使用的。 对于脑组织,我们使用朊病毒蛋白,它也存在于脂筏中

食谱

  1. 裂解缓冲液
    150mM NaCl 25mM Tris-HCl(pH7.4) 5 mM EDTA
    1%Triton X-100 过滤灭菌并保持在4°C
  2. 渐变缓冲区
    150mM NaCl 25mM Tris-HCl(pH7.4) 5 mM EDTA
    过滤灭菌并保持在4°C

致谢

该协议改编自Lemaire-Vieille等人(2013)。

参考文献

  1. Aboulaich,N。(2011)。 从含洗涤剂的样品中沉淀蛋白。 生物协议 1(5 ):e40。 
  2. Lemaire-Vieille,C.,Bailly,Y.,Erlich,P.,Loeuillet,C.,Brocard,J.,Haeberle,AM,Bombarde,G.,Rak,C.,Demais,V.,Dumestre-Perard, C.,Gagnon,J。和Cesbron,JY(2013)。 在表达嵌合PrP-Dpl蛋白的小鼠中具有小脑损伤的共济失调。 Neurosci 33(4):1391-1399。
  3. Persaud-Sawin,D.A.,Lightcap,S。和Harry,G.J。(2009)。 通过无洗涤剂方法从小鼠脑组织中分离筏。 J Lipid Res 50(4):759-767。
  • English
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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. Lemaire-Vieille, C., Gagnon, J. and Cesbron, J. (2013). Protein Flotation Assay to Isolate Lipids Rafts from Soft Tissue or Cells. Bio-protocol 3(16): e854. DOI: 10.21769/BioProtoc.854.
  2. Lemaire-Vieille, C., Bailly, Y., Erlich, P., Loeuillet, C., Brocard, J., Haeberle, A. M., Bombarde, G., Rak, C., Demais, V., Dumestre-Perard, C., Gagnon, J. and Cesbron, J. Y. (2013). Ataxia with cerebellar lesions in mice expressing chimeric PrP-Dpl protein. J Neurosci 33(4): 1391-1399.
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