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Preparation of Golgi Membranes from Rat Liver
从大鼠肝中制备高尔基体膜   

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Abstract

This protocol details the isolation of enriched Golgi membranes from rat liver, using discontinuous density gradient centrifugation. This high-yield extraction method is useful for several applications, including immunoprecipitation of solubilised Golgi membrane proteins (preparation included) and electron microscopy. Protocol adapted from Leelavathi et al. (1970).

Keywords: Golgi membranes(高尔基体膜), Density gradient centrifugation(密度梯度离心), Solubilised Golgi membrane proteins(高尔基体膜蛋白的可溶性), Rat liver(建议肝), Tissue isolation(组织分离)

Materials and Reagents

  1. Fresh rat liver tissue (30 mg)
  2. HEPES (Sigma-Aldrich, catalog number: H4034 )
  3. KCl (Baker, catalog number: 0 208 )
  4. Triton X-100 (ACROS, catalog number: 215680010 )
  5. EDTA (Baker, catalog number: 1073 )
  6. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  7. β-Glycerophosphate (Sigma-Aldrich, catalog number: G9422 )
  8. NaF (Sigma-Aldrich, catalog number: S7920 )
  9. Protease inhibitor cocktail (complete Mini EDTA-free) (Roche Diagnostics; or equivalent) (Roche Diagnostics, catalog number: 0 5056489001 )
  10. Protein assay kit (Bio-Rad Laboratories, catalog number: 500-0006 )
  11. MgCl2 (Sigma-Aldrich, catalog number: 16104 )
  12. Sucrose (Sigma-Aldrich, catalog number: 16104; extra pure grade) (see Recipes)
  13. Phosphatase inhibitor cocktail (Roche Diagnostics, catalog number: 04906837001 ) (see Recipes)

Equipment

  1. Ultraturrax homogeniser (T 25 digital ULTRA-TURRAX® IKA® Werke Staufen/Germany)
  2. Centrifuge (Ultracentrifuge, Beckman Coulter, model: Optima XE-100; ultra clear centrifuge tubes, catalog number: 344058 )
  3. SW28 (or equivalent) swing-out rotor [swinging bucket rotor, catalog number: 2352 ; Beckman SW 28 (Beckman Coulter, catalog numbers: 342207 , 342204 ), with titanium buckets 6 x 38.5 ml, 25 x 89 mm, 28,000 rpm, 141,000 x g]
  4. Bausch and Lomb refractometer (Lab Logistics Group GmbH)

Procedure

  1. About 30 mg rat liver tissue is excised from six male Sprague-Dawley rats (weighing 200-300 g each) fed ad libitum the day before the Golgi-membrane preparation. The tissue is placed into 100 ml ice-cold 0.5 M sucrose solution and squeezed to expel the blood. Then, the tissue is minced to a fine purée with scissors in 140 ml ice-cold 0.5 M sucrose solution, and then homogenised for 30 sec at 12 Watt using an Ultraturrax homogeniser (or equivalent) at 4 °C.
  2. The homogenate is centrifuged for 10 min at 600 x g at 4 °C.
  3. The supernatant (~120 ml in 0.5 M ice-cold sucrose solution) is then split into 20 ml aliquots, each of which is slowly layered onto an 18 ml 1.3 M sucrose cushion by being carefully dripped down the side of the 38.5 ml centrifuge tubes (so as not to disturb the cushion) for the SW28 (or equivalent) swing-out rotor. Centrifuge the cushion plus homogenate supernatant for 60 min at 105,000 x g at 4 °C in an SW28 swing-out rotor. This results in the formation of: (a) an upper fat layer; (b) a clear reddish supernatant; (c) a thick membrane felt layer above the 1.3 M sucrose interface; (d) a turbid 1.3 M sucrose supernatant; and (e) a sediment at the bottom of the tube.
  4. The interface between the 1.3 M and 0.5 M sucrose layers (~2.5 ml per tube) is collected and used for the preparation of the Golgi-rich fraction. This is collected by carefully puncturing the tube at the middle of the interface with an 18 gauge needle (3.8 cm long) and using a syringe to extract the interface. This is gently resuspended and the molarity of the sucrose adjusted to 1.1 M by adding 2.3 M sucrose (usually to a final volume of ~16 ml), with the aid of a Bausch and Lomb (or equivalent) refractometer. The volume is then brought to a total of 18 ml with 1.1 M sucrose.
  5. The suspension is split into two 9-ml aliquots that are layered onto sucrose gradients that are previously prepared using 7.5 ml each of 1.4 M, 1.3 M and then 1.25 M sucrose, layered on top of each other from the bottom of the cellulose acetate tubes (using the same tubes as in step 3) for the SW28 (or equivalent) swing-out rotor. Finally, the 1.1 M sucrose sample layer is covered with 6.5 ml 0.5 M sucrose solution. These stages are all carried out with care taken not to disturb or mix the layers of the gradient in any way. Prior to the layering of the sample onto the 1.25 M sucrose, the prepared gradients are stored for at least 20 min at 4 °C.
  6. These are centrifuged for 90 min at 105,000 x g at 4 °C, and then the Golgi-enriched membrane fraction is collected at the interface between the 0.5 M and 1.1 M sucrose (as for step 4).
  7. The collected Golgi-enriched membrane fractions are pooled and assayed for protein concentration using a commercially available protein assay kit, according to the manufacturer instructions. This provides an estimate of the yield of the Golgi fraction and its contamination with rough endoplasmic reticulum membranes and/or free ribosomes. The yield for the Golgi-enriched fraction is about 1.5 mg to 2.0 mg of protein per 1 g liver. The pooled Golgi-enriched membrane fractions are then aliquoted as required, frozen in liquid nitrogen, and stored at -80 °C. Routinely, for batch control variations, Western blotting analysis of the Golgi-enriched fraction protein is performed using antibodies against Golgi-localised and/or endoplasmic reticulum-localised proteins (e.g., anti-mannosidase and anti-protein disulphide isomerase antibodies, respectively).
  8. To solubilise these Golgi-enriched membranes for use in pull-down assays, 4 mg Golgi-enriched membrane proteins (800 μl) are diluted two-fold (1:1) in 10 mM HEPES (pH 8.0), 50 mM KCl, with protease and phosphatase inhibitor cocktails added, centrifuged (10 min, 20,000 x g, 4 °C). This wash step is then repeated again, resuspending the Golgi-enriched membrane pellets in 800 μl of the same buffer.
  9. The final Golgi-enriched membrane pellet is resuspended in 400 μl 20 mM HEPES (pH 8.0), 100 mM KCl, 1% Triton X-100, 2 mM EDTA, with protease and phosphatase inhibitor cocktails added, and solubilised for 30 min at 4 °C.
  10. The solubilised suspension is then diluted 1:1 with water (to a final 800 μl volume), and centrifuged for 10 min at 2,000 x g at 4 °C. The supernatant from this final centrifugation is used as the “solubilised Golgi-enriched membranes”.

Recipes

  1. Sucrose
    Sucrose solutions (0.5 M, 1.1 M, 1.25 M, 1.3 M, 1.4 M, 2.3 M) in 0.1 M potassium phosphate buffer (pH 6.65) (combine 38.1 ml of 1 M K2HPO4 stock solution with 61.9 ml of 1 M KH2PO4 stock solution and then dilute to 1 L with distilled H2O) with 5 mM MgCl2
  2. Phosphatase inhibitor cocktail
    1 μM sodium orthovanadate
    20 mM β-glycerophosphate
    10 mM NaF
    This cocktail is made up in water at 4 °C, immediately before use.

Acknowledgments

The authors would like to thank: The Italian Association for Cancer Research (to D. C. IG4664, IG10341; to A. L. IG4700), Telethon Italia (to D. C. GGPO9274; to A. L. GGPO8231), the European Community Seventh Framework Programme FP7/2007-2013 HEALTH-F2-2007-201804 (Eucilia to A. L.), grant FIT DM 24/09/2009, Legge 46/82, Ministry of Economy and Finance (to D. C.), PON project 01/00117 2007-2013 “Antigens and adjuvants for vaccines and immunotherapy” and the MIUR Project “FaReBio di Qualità”, for financial support. C. V. and S. S. were recipients of Italian Foundation for Cancer Research (FIRC, Milan, Italy) Fellowships. This protocol was adapted from Leelavathi et al. (1970), as detailed in Valente et al. (2012). The authors also thank C.P. Berrie for editorial assistance.

References

  1. Leelavathi, D., Estes, L., Feingold, D. S. and Lombardi, B. (1970). Isolation of a Golgi-rich fraction from rat liver. Biochim Biophys Acta 211(2): 124-138.
  2. Valente, C., Turacchio, G., Mariggio, S., Pagliuso, A., Gaibisso, R., Di Tullio, G., Santoro, M., Formiggini, F., Spano, S., Piccini, D., Polishchuk, R. S., Colanzi, A., Luini, A. and Corda, D. (2012). A 14-3-3gamma dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIbeta to regulate post-Golgi carrier formation. Nat Cell Biol 14(4): 343-354.

简介

该协议详述了使用不连续密度梯度离心从大鼠肝脏分离富集的高尔基体膜。 这种高产率提取方法可用于几种应用,包括溶解的高尔基膜蛋白(包括制备)和电子显微镜的免疫沉淀。 从Leelavathi等人改编的方案(1970)。

关键字:高尔基体膜, 密度梯度离心, 高尔基体膜蛋白的可溶性, 建议肝, 组织分离

材料和试剂

  1. 新鲜大鼠肝组织(30mg)
  2. HEPES(Sigma-Aldrich,目录号:H4034)
  3. KCl(Baker,目录号:0208)
  4. Triton X-100(ACROS,目录号:215680010)
  5. EDTA(Baker,目录号:1073)
  6. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
  7. β-甘油磷酸酯(Sigma-Aldrich,目录号:G9422)
  8. NaF(Sigma-Aldrich,目录号:S7920)
  9. 蛋白酶抑制剂混合物(完全Mini无EDTA)(Roche Diagnostics;或等效物)(Roche Diagnostics,目录号:05056489001)
  10. 蛋白质测定试剂盒(Bio-Rad Laboratories,目录号:500-0006)
  11. MgCl 2(Sigma-Aldrich,目录号:16104)
  12. 蔗糖(Sigma-Aldrich,目录号:16104;超纯级)(参见Recipes)
  13. 磷酸酶抑制剂混合物(Roche Diagnostics,目录号:04906837001)(参见Recipes)

设备

  1. Ultraturrax均化器(T25数字ULTRA-TURRAX IKA Werke Staufen/Germany)
  2. 离心机(超速离心机,Beckman Coulter,型号:Optima XE-100;超清离心管,目录号:344058)
  3. SW28(或等效)摆出式转子[摆动斗式转子,目录号:2352; Beckman SW 28(Beckman Coulter,目录号:342207,342204),具有6×38.5ml,25×89mm,28,000rpm,141,000×g的钛桶
  4. Bausch和Lomb折光计(Lab Logistics Group GmbH)

程序

  1. 在高尔基膜制备前一天自由进食的六只雄性Sprague-Dawley大鼠(每只重200-300g)中摘出约30mg大鼠肝组织。 将组织置于100ml冰冷的0.5M蔗糖溶液中并挤压以排出血液。 然后,用剪刀在140ml冰冷的0.5M蔗糖溶液中将组织切碎成细粉,然后使用Ultraturrax匀浆器(或等同物)在4℃下在12瓦特下匀化30秒。
  2. 匀浆在4℃以600×g离心10分钟。
  3. 然后将上清液(在0.5M冰冷的蔗糖溶液中〜120ml)分成20ml等分试样,将每一等分试样缓慢地分层到18ml 1.3M蔗糖缓冲液上,将其小心地滴在38.5ml 离心管(以免干扰缓冲垫)用于SW28(或等效的)旋出式转子。在SW28转出转子中,在4℃下,以105,000×g离心缓冲液加匀浆上清液60分钟。这导致形成:(a)上部脂肪层; (b)澄清的红色上清液; (c)在1.3M蔗糖界​​面上方的厚膜毡层; (d)混浊的1.3M蔗糖上清液;和(e)在管底部的沉淀物
  4. 收集1.3M和0.5M蔗糖层之间的界面(每管约2.5ml),并用于制备富含高尔基体的级分。通过使用18号针头(3.8cm长)在界面中间小心地刺破管并使用注射器提取界面来收集。通过Bausch和Lomb(或等效的)折射计的帮助,通过加入2.3M蔗糖(通常至〜16ml的最终体积)将其轻轻地重悬,并将蔗糖的摩尔浓度调节至1.1M。然后用1.1M蔗糖使体积达到总共18ml。
  5. 将悬浮液分成两个9-ml等分试样,将其分层到蔗糖梯度上,所述蔗糖梯度预先使用7.5ml 1.4M,1.3M和1.25M蔗糖制备,并从乙酸纤维素管的底部彼此层叠(使用与步骤3相同的管)用于SW28(或等效的)摆出转子。最后,用6.5ml 0.5M蔗糖溶液覆盖1.1M蔗糖样品层。这些阶段全部进行,注意不以任何方式干扰或混合梯度的层。在将样品层合到1.25M蔗糖上之前,将制备的梯度在4℃下储存至少20分钟。
  6. 将这些在4℃下以105,000×g离心90分钟,然后在0.5M至1.1M蔗糖之间的界面处收集高尔基体富集的膜级分(如步骤4所示)。
  7. 收集收集的富集高尔基体的膜级分,并使用市售的蛋白质测定试剂盒,根据制造商的说明书测定蛋白质浓度。这提供了高尔基部分的产量的估计及其对粗糙的内质网膜和/或游离核糖体的污染。富含高尔基体的级分的产量为每1g肝脏约1.5mg至2.0mg蛋白质。合并的Golgi然后根据需要将富集的膜级分等分,在液氮中冷冻,并储存在-80℃。通常,对于批次对照变异,使用针对高尔基体定位的和/或内质网定位的蛋白质的抗体(例如抗 - 甘露糖苷酶和抗蛋白质)进行高尔基体富集的部分蛋白质的蛋白质印迹分析二硫化物异构酶抗体)
  8. 为了溶解这些用于下拉测定的高尔基体膜,将4mg高尔基体膜蛋白(800μl)在10mM HEPES(pH 8.0),50mM KCl中稀释两倍(1:1),加入蛋白酶和磷酸酶抑制剂混合物,离心(10分钟,20,000×g,4℃)。然后再次重复该洗涤步骤,将富含高尔基体的膜沉淀重悬在800μl的相同缓冲液中。
  9. 将最终的高尔基体膜沉淀重悬于400μl20mM HEPES(pH 8.0),100mM KCl,1%Triton X-100,2mM EDTA中,加入蛋白酶和磷酸酶抑制剂混合物,并在4℃溶解30分钟C。
  10. 然后将溶解的悬浮液用水以1:1稀释(至最终800μl体积),并在4℃以2,000xg离心10分钟。来自该最终离心的上清液用作"溶解的高尔基体膜"

食谱

-
  1. 蔗糖
    在0.1M磷酸钾缓冲液(pH 6.65)中的蔗糖溶液(0.5M,1.1M,1.25M,1.3M,1.4M,2.3M)(组合38.1ml的1MK sub 2 HPO, 4升储备溶液与61.9ml 1M KH 2 PO 4 /储备溶液混合,然后用蒸馏H 2 SO 4稀释至1L, O)与5mM MgCl 2·h/v
  2. 磷酸酶抑制剂混合物
    1μM原钒酸钠
    20mMβ-甘油磷酸盐 10mM NaF 这种鸡尾酒在4°C的水中配制,在使用前

致谢

作者要感谢:意大利癌症研究协会(对D. C. IG4664,IG10341;对A. L. IG4700),Telethon Italia(对D. C. GGPO9274;对A. L. GGPO8231),欧洲共同体第七框架计划 FP7/2007-2013 HEALTH-F2-2007-201804(Eucilia to AL),授予FIT DM 24/09/2009,Legge 46/82,经济和财政部(DC),PON项目01/00117 2007-2013 "疫苗和免疫治疗的抗原和佐剂"和MIUR项目"FaReBio diQualità",用于资金支持。 C. V.和S. S.获得意大利癌症研究基金会(FIRC,米兰,意大利)奖学金。该方案改编自Leelavathi等人(1970),如Valente等人(2012)中详细描述的。作者还感谢C.P. Berrie提供编辑协助。

参考文献

  1. Leelavathi,D.,Estes,L.,Feingold,D.S。和Lombardi,B。(1970)。 从大鼠肝中分离富含高尔基体的部分。生物化学生物物理学 211(2):124-138。
  2. Valente,C.,Turacchio,G.,Mariggio,S.,Pagliuso,A.,Gaibisso,R.,Di Tullio,G.,Santoro,M.,Formiggini,F.,Spano,S.,Piccini, ,Polishchuk,RS,Colanzi,A.,Luini,A.and Corda,D。(2012)。 A 14-3-3基于二聚体的支架将CtBP1-S/BARS桥接到PI(4) KIIIbeta以调节高尔基体载体形成。 Nat Cell Biol 14(4):343-354。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Valente, C., Turacchio, G., Spanò, S., Luini, A. and Corda, D. (2014). Preparation of Golgi Membranes from Rat Liver. Bio-protocol 4(22): e1291. DOI: 10.21769/BioProtoc.1291.
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