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In vitro Lipid Transfer Assay
体外脂质转移试验

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Abstract

This is a protocol to detect lipid transfer activity of NRF-5, a member of the LPS binding/lipid transfer protein family. The lipid transfer activity is examined by using isotope-labeled cholesterol and liposomes, and tested in two directions (Figure 1): from proteins to liposomes and from liposomes to proteins.

Materials and Reagents

  1. PC (Avanti-Polar Lipids)
  2. PE (Avanti-Polar Lipids)
  3. Cholesterol (Avanti-Polar Lipids, catalog number: 700000 )
  4. 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) (Avanti, catalog number: 850725 )
  5. 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC) (Avanti, catalog number: 770375 )
  6. 1 mCi (37 MBq) (PerkinElmer,  catalog number: NET139001MC )
  7. Protein of interest tagged with Flag
  8. Tris-HCl (pH 7.4)
  9. NaCl
  10. Anti-Flag M2 agarose beads (Sigma-Aldrich, catalog number: A2220 )
  11. Flag peptide (Sigma-Aldrich, catalog number: F3290 )
  12. Chloroform
  13. Wash buffer (see Recipes)
  14. Elution buffer (see Recipes)

Equipment

  1. Avanti Mini-Extruder (Avanti, catalog number: 610023 )
  2. Vortexer
  3. Centrifuges
  4. Rotator
  5. Branson tip-sonicator (Cole-parmer Cp750)
  6. Scintillation counter (Wallac MicroBeta TriLux, catalog number: 1450-023 )

Procedure

  1. Preparation of liposomes (room temperature)
    1. Liposome in the absence of cholesterol is made by Avanti Mini-Extruder at room temperature. The dried 1.25 mg mix of PC (75%) and PE (25%) is hydrated in 1 ml buffer (50 mM Tris-Cl, 150 mM NaCl). 100 nm unilamellar vesicles are obtained by extrusion as described:
      (http://www.avantilipids.com/index.php?option=com_content&view=article&id=185&Itemid=193)
    2. Liposomes containing [3H]cholesterol are generated by using a standard sonication procedure.
      1. Dissolve dried 2.5 mg mix of PC (75%) and PE (25%) in 200 μl chloroform by vortex.
      2. Take 8 μl of above chloroform dissolved lipid mix and add 0.001 mg [3H]cholesterol (~2% molar mass).
      3. The chloroform is evaporated and dried under a stream of nitrogen. Longer drying time (4-12 h) can be used to remove any trace of organic solvent.
      4. The dry lipid film is hydrated by adding 0.5 ml of buffer (50 mM Tris-Cl, 150 mM NaCl). After vortex at room temperature for 20 min, the large multilamellar vesicle suspension is disrupted with a Branson tip-sonicator until the suspension clear. For sonication, the samples are placed on ice and sonicated for 10 min with cycles including 9 seconds sonication, 9 seconds interval and 35% input.
      5. Metal particles from the sonicator tip and undisrupted lipid aggregates are removed by centrifugation at 100,000 x g for 30 min at 4 °C. The resulting hazy supernatant, composed primarily of small unilamellar vesicles, is stored at 4 °C. The liposomes can be stored at this condition for one week.
  2. Examine [3H]Cholesterol transfer from liposomes to proteins (Figure 1)
    Reactions are performed on ice.
    1. In the [3H]cholesterol/liposome to protein transfer assay, each reaction contains, in a final volume of 200 μl buffer (50 mM Tris-Cl, 150 mM NaCl, pH 7.4), 4 μg of PC:PE:[3H]cholesterol liposomes, and different amounts of the acceptor protein EGFP-FLAG and EGFP::NRF-5-FLAG(15 and 30 μg) purified from 293T cells (Zhang et al., 2012). EGFP-FLAG is used as the negative control.
    2. After incubation for 30 min at 4 °C, each mixture is diluted with 600 μl 50 mM Tris-Cl, 150 mM NaCl, followed by adding 70 μl 50% Flag beads.
    3. After incubation for about 1 h at 4 °C on shaker, the Flag beads are washed 4-5 times in 1,000 μl wash buffer on shaker, and bounded [3H]cholesterol is quantified by scintillation counting.


      Figure 1. Schematic diagrams of the lipid transfer assay in two directions

  3. Examine [3H]Cholesterol transfer from proteins to liposomes (Figure 1)
    1. Protein-[3H]cholesterol complex is obtained by incubating EGFP-FLAG or EGFP::NRF-5-FLAG (400 pmol) with [3H]cholesterol (100 pmol) in a final volume of 300 μl buffer (50 mM Tris-Cl, 150 mM NaCl, pH 7.4) for 3 h at 4 °C. The protein-[3H]cholesterol complex is pulled down by incubating with 100 μl 50% Flag beads for 2 h at 4 °C, washing 6 times as above and eluted with 100 μl Flag peptide (100 mg ml-1) for two times.
    2. In the protein-to-liposome transfer assay, each reaction contains, in a final volume of 200 μl buffer (50 mM Tris-Cl, 150 mM NaCl, pH 7.4), [3H]cholesterol complexed to either EGFP or EGFP-NRF-5 (40 μl), and different amounts of acceptor PC liposomes (50 and 100 ng).
    3. After incubation for 30 min at 4 °C, each mixture is diluted with 600 μl of buffer (50 mM Tris-Cl, 150 mM NaCl, pH 7.4).
    4. Liposomes are separated by centrifuging at 10,000 x g (hard to detect weight at this stage) for 30 min at 4 °C. The lipososomes can be seen as a small white patch at the bottom of the tube.
    5. Wash the liposomes 4-5 times in the buffer (50 mM Tris-HCl, 150 mM NaCl), with 500 μl buffer used in each tube at each time. The liposomes are collected by centrifuge (10,000 x g) after each wash. The amount of [3H]cholesterol transferred to liposomes is determined by scintillation counting.

Recipes

  1. Wash buffer
    50 mM Tris-Cl, 150 mM NaCl (pH 7.4)
  2. Elution buffer
    50 mM Tris-Cl, 150 mM NaCl (pH 7.4), Flag peptide

Acknowledgments

This protocol is adapted from Zhang et al. (2012) and Infante et al. (2008).

References

  1. Infante, R. E., Wang, M. L., Radhakrishnan, A., Kwon, H. J., Brown, M. S. and Goldstein, J. L. (2008). NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci U S A 105(40): 15287-15292.
  2. Zhang, Y., Wang, H., Kage-Nakadai, E., Mitani, S. and Wang, X. (2012). C. elegans secreted lipid-binding protein NRF-5 mediates PS appearance on phagocytes for cell corpse engulfment. Curr Biol  22(14): 1276-1284.

简介

这是一种检测NRF-5(LPS结合/脂质转移蛋白家族成员)的脂质转移活性的方案。 通过使用同位素标记的胆固醇和脂质体检查脂质转移活性,并在两个方向(图1)测试:从蛋白质到脂质体,从脂质体到蛋白质。

材料和试剂

  1. PC(Avanti-Polar Lipids)
  2. PE(Avanti-Polar Lipids)
  3. 胆固醇(Avanti-Polar Lipids,目录号: 700000)
  4. 1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)(Avanti ,目录号: span> 850725)
  5. 1,2-二 - (9Z-十八烯酰基)-sn-甘油基-3-胆碱磷酸(DOPC)(Avanti , ; number: 770375)
  6. 1 mCi(37MBq)(PerkinElmer,目录号:NET139001MC)
  7. 标记为Flag的利益蛋白质
  8. Tris-HCl(pH 7.4)
  9. NaCl
  10. 将抗Flag M2琼脂糖珠(Sigma-Aldrich,目录号: A2220)
  11. Flag肽(Sigma-Aldrich,目录号: F3290)
  12. 氯仿
  13. 洗涤缓冲液(见配方)
  14. 洗脱缓冲液(见配方)

设备

  1. Avanti迷你挤出机(Avanti,目录号:610023)
  2. Vortexer
  3. 离心机
  4. 旋转器
  5. Branson tip-sonicator(Cole-parmer Cp750)
  6. 闪烁计数器(Wallac MicroBeta TriLux,目录号:1450-023)

程序

  1. 脂质体的制备(室温)
    1. 在不存在胆固醇的情况下的脂质体通过Avanti Mini-Extruder在室温下制备。干燥的1.25mg PC(75%)和PE(25%)混合物在1ml缓冲液(50mM Tris-Cl,150mM NaCl)中水合。通过如所述的挤出获得100nm单层囊泡:
      http://www.avantilipids.com/index.php? option = com_content& view = article& id = 185& Itemid = 193
    2. 含有[3 H]胆固醇的脂质体通过使用标准超声处理程序产生。
      1. 通过涡旋溶解干燥2.5mg PC(75%)和PE(25%)在200μl氯仿中的混合物。
      2. 取8μl上述氯仿溶解的脂质混合物,并加入0.001mg [3 H]胆固醇(〜2%摩尔质量)。
      3. 蒸发氯仿并在氮气流下干燥。较长的干燥时间(4-12小时)可用于除去任何痕量的有机溶剂。
      4. 通过加入0.5ml缓冲液(50mM Tris-Cl,150mM NaCl)使干脂质膜水合。在室温下涡旋20分钟后,用Branson尖端超声仪破碎大的多层囊泡悬浮液直到悬浮液澄清。对于超声处理,将样品置于冰上并用包括9秒超声,9秒间隔和35%输入的循环超声处理10分钟。
      5. 来自超声波发生器尖端的金属颗粒和未破坏的脂质聚集体通过在4℃下以100,000×1000g离心30分钟而去除。将所得的主要由小单层囊泡组成的混浊上清液在4℃下储存。脂质体可以在这种条件下储存一周。
  2. 检查[脂质体]胆固醇从脂质体转移到蛋白质(图1) 反应在冰上进行。
    1. 在[3 H]胆固醇/脂质体至蛋白质转移测定中,每个反应在最终体积为200μl缓冲液(50mM Tris-Cl,150mM NaCl,pH 7.4)中含有4μg的PC:PE:[3 H]胆固醇脂质体,以及从293T细胞纯化的不同量的受体蛋白EGFP-FLAG和EGFP :: NRF-5-FLAG(15和30μg) et al。,2012)。 EGFP-FLAG用作阴性对照
    2. 在4℃下孵育30分钟后,每种混合物用600μl50mM Tris-Cl,150mM NaCl稀释,然后加入70μl50%Flag珠。
    3. 在振荡器上在4℃下孵育约1小时后,将Flag珠在摇动器上在1,000μl洗涤缓冲液中洗涤4-5次,通过闪烁计数定量结合的[3 H]胆固醇。


      图1.两个方向的脂质转移测定示意图

  3. 检测胆固醇从蛋白质转移到脂质体(图1)
    1. 通过将EGFP-FLAG或EGFP :: NRF-5-FLAG(400pmol)与[3 H]胆固醇(100μL)一起温育获得蛋白质 - [3 H]胆固醇复合物pmol)在终体积300μl缓冲液(50mM Tris-Cl,150mM NaCl,pH7.4)中在4℃温育3小时。通过在4℃下与100μl50%Flag珠孵育2小时,将蛋白质 - [3 H]胆固醇复合物下拉,洗涤6次,并用100μlFlag肽(100μl)洗脱, mg ml -1 )两次。
    2. 在蛋白质 - 脂质体转移测定中,每个反应在最终体积为200μl缓冲液(50mM Tris-Cl,150mM NaCl,pH7.4)中含有[3 H]胆固醇复合物到EGFP或EGFP-NRF-5(40μl)和不同量的受体PC脂质体(50和100ng)。
    3. 在4℃下培养30分钟后,每种混合物用600μl缓冲液(50mM Tris-Cl,150mM NaCl,pH7.4)稀释。
    4. 通过在4℃下以10,000×g(难以在此阶段检测重量)离心30分钟来分离脂质体。脂质体可以看作是管底部的小白色斑点
    5. 在缓冲液(50mM Tris-HCl,150mM NaCl)中洗涤脂质体4-5次,每次使用在每个管中的500μl缓冲液。在每次洗涤后通过离心收集脂质体(10,000×g/g)。转移到脂质体中的[3 H]胆固醇的量通过闪烁计数测定。

食谱

  1. 洗涤缓冲液
    50mM Tris-Cl,150mM NaCl(pH7.4)
  2. 洗脱缓冲液
    50mM Tris-Cl,150mM NaCl(pH 7.4),Flag肽

致谢

该协议改编自Zhang等人(2012)和Infante等人(2008)。

参考文献

  1. Infante,R.E.,Wang,M.L.,Radhakrishnan,A.,Kwon,H.J.,Brown,M.S.and Goldstein,J.L。(2008)。 NPC2促进胆固醇在NPC1和脂质双层之间的双向转移,胆固醇从溶酶体中排出的步骤。 Proc Natl Acad Sci U S A 105(40):15287-15292。
  2. Zhang,Y.,Wang,H.,Kage-Nakadai,E.,Mitani,S.and Wang,X.(2012)。 C 。 eleg ans 分泌的脂质结合蛋白NRF-5介导吞噬细胞上的PS表现,用于细胞吞噬。
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引用:Zhang, Y. and Wang, X. (2013). In vitro Lipid Transfer Assay. Bio-protocol 3(9): e691. DOI: 10.21769/BioProtoc.691.
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