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Subcellular Fractionation Using Accudenz Gradient to Separate ER/Golgi in Yeast
使用Accudenz细胞组分梯度离心法分离酵母中的ER/Golgi   

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Abstract

This protocol describes how to separate the endoplasmic reticulum (ER) and Golgi apparatus in yeast cells using a subcellular fractionation approach with an Accudenz gradient.

Materials and Reagents

  1. Accudenz (Accurate Chemical & Scientific Corporation)
  2. Protease inhibitors
    1. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 78830-5G )
    2. Aprotinin (Sigma-Aldrich, catalog number: A3428-10MG )
    3. Pepstatin A (Sigma-Aldrich, catalog number: P5318-5MG )
  3. NaN3
  4. Sodium fluoride (NaF)
  5. Tris-HCl
  6. Beta-mercaptoethanol
  7. TCA
  8. Sorbitol
  9. HEPES-KOH
  10. Spheroplasting buffer (see Recipes)
  11. Lysis buffer (see Recipes)

Equipment

  1. Dounce homogenizer (Cole-Parmer)
  2. Refractometer (Bausch & Lomb Incorporated)
  3. Beckman rotor

Procedure

  1. Preparation of cell lysate
    1. Collect 50 OD of cells and wash once in 10 mM NaN3, 1 mM sodium fluoride (NaF), 50 mM Tris-HCl (pH 7.5), to poison energy-dependent processes.
    2. Incubate them in 10 OD/ml 100 mM Tris-HCl (pH 9.4), 50 mM beta-mercaptoethanol, 10 mM NaN3 at room temperature (RT) for 10 min to reduce disulfide bonds in the cell wall.
    3. Wash and resuspend in Spheroplasting buffer 30 ~50 OD/ml and incubate at 37 °C until 90% of the cells are converted to spheroplasts (30 ~ 40 min).
    4. Centrifuge at 3,000 x g for 5 min to collect spheroplasts.
    5. Resuspend in 2 ml of Lysis buffer. Lyse cells in a dounce homogenizer (tight pestle, 15 strokes).
    6. Centrifuge lysates at 500 x g for 5 min to clear unbroken cells. Centrifuge twice if necessary.

  2. Preparation of Accudenz gradient
    1. Prepare gradient solutions in lysis buffer. Generate gradients with the following weight/volume amounts of Accudenz: 1 ml 43%, 1 ml 37%, 1 ml 31%, 1.5 ml 27%, 1.5 ml 23%, 1.5 ml 20%, 1 ml 17%, 1 ml 13% and 1 ml 8%.
    2. Measure the refractive index of the standard Accudenz gradient using a refractometer and convert these values to grams per milliliter based on a standard curve generated by five weighed standards.
    3. Measure the refractive index of the collected fractions to determine their densities.

  3. Fractionation by equilibrium sedimentation
    1. Intracellular membranes can be separated on the basis of their characteristic densities, and cofractionation of the protein of interest with a known membrane marker protein can be examined.
    2. Load the cleared lysates at the top of the Accudenz gradient (8% - 43%) and centrifuge to equilibrium in a Beckman rotor for 18 h at 170,000 x g at 4 °C. Use slow break.
    3. Collect 12 fractions from the top of the gradient; precipitate proteins with 10% TCA.

  4. Western blot
    Catalog number or source
    (MP biomedicals)
    Yeast gene name
    Yeast antigen tecognized by antibody
    Yeast organelle in which antigen resides
    Monoclonal or polyclonal, host
    Western blots
    (g/ml)


    A-6427
    Vma 2
    V-ATPase 60 kDa subunit
    Vacuole membranes


    A-6429
    Dpm 1
    Dol-P-Man Synthase
    ER


    A-6457
    PGK
    3-Phosphoglycerate
    Cytoplasm




    Kinase




Recipes

  1. Spheroplasting buffer
    Spheroplasting buffer
    100 ml
    200 ml
    1 M sorbitol (FW 182.17)


    10 mM NaN3


    10 μg/μl oxolyticase or 20~40 U/OD oxolyticase


    40 mM HEPES-KOH (pH 7.5)



  2. Lysis buffer
    Lysis buffer
    100 ml
    200 ml
    0.2 M sorbitol (FW 182.17)


    50 mM KOAc


    2 mM EDTA


    20 mM HEPES-KOH (pH 6.8)



  3. Add protease inhibitors to final concentration (20 μg/ml PMSF, 5 μg/ml antipain, 1 μg/ml each of aprotinin, leupeptin, and pepstatin, and 10 μg/ml alpha2-marcroglobulin).

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

Reference

  1. Cowles, C. R., Odorizzi, G., Payne, G. S. and Emr, S. D. (1997). The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole. Cell 91(1): 109-118.

简介

该协议描述了如何使用具有Accudenz梯度的亚细胞分级方法在酵母细胞中分离内质网(ER)和高尔基体。

材料和试剂

  1. Accudenz(Accurate Chemical& Scientific Corporation)
  2. 蛋白酶抑制剂
    1. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:78830-5G)
    2. 抑肽酶(Sigma-Aldrich,目录号:A3428-10MG)
    3. 胃酶抑素A(Sigma-Aldrich,目录号:P5318-5MG)
  3. NaN 3
  4. 氟化钠(NaF)
  5. Tris-HCl
  6. β-巯基乙醇
  7. TCA
  8. 山梨醇
  9. HEPES-KOH
  10. 球形缓冲液(见配方)
  11. 裂解缓冲液(见配方)

设备

  1. Dounce匀浆器(Cole-Parmer)
  2. 折射计(Bausch& Lomb Incorporated)
  3. Beckman转子

程序

  1. 细胞裂解液的制备
    1. 收集50OD的细胞并在10mM NaN 3,1mM氟化钠(NaF),50mM Tris-HCl(pH7.5)中洗涤一次以中毒能量依赖性过程。
    2. 将它们在室温(RT)下在10OD/ml 100mM Tris-HCl(pH 9.4),50mMβ-巯基乙醇,10mM NaN 3中孵育10分钟以还原 细胞壁
    3. 洗涤并重悬于球形造粒缓冲液30〜50 OD/ml,并在37℃孵育直至90%的细胞转化为原生质球(30〜40分钟)。
    4. 在3,000×g离心5分钟以收集原生质球。
    5. 重悬于2ml裂解缓冲液中。 在dounce匀浆器中裂解细胞(紧杵,15次)
    6. 离心裂解物在500×g离心5分钟以清除未破碎的细胞。 如有必要,离心两次。

  2. 制备Accudenz梯度
    1. 准备在裂解缓冲液中的梯度溶液。 用以下重量/体积量的Accudenz产生梯度:1ml 43%,1ml 37%,1ml 31%,1.5ml 27%,1.5ml 23%,1.5ml 20%,1ml 17%,1ml 13 %和1ml 8%
    2. 使用折射计测量标准Accudenz梯度的折射率,并基于由五个称重的标准物产生的标准曲线将这些值转换成克/毫升。
    3. 测量收集的馏分的折射率,以确定它们的密度
  3. 通过平衡沉降分馏
    1. 可以基于它们的特征密度分离细胞内膜,并且可以检查目的蛋白质与已知膜标记蛋白质的共分级。
    2. 将澄清的裂解物装载在Accudenz梯度(8%-43%)的顶部,并在Beckman转子中在4℃以170,000×g离心18小时。 使用缓慢中断。
    3. 从梯度的顶部收集12个馏分; 用10%TCA沉淀蛋白质。

  4. 蛋白质印迹
    目录号码或来源
    (MP生物医学)
    酵母基因名称
    抗体识别的酵母抗原
    抗原所在的酵母细胞器
    单克隆或多克隆,宿主
    Western blots
    (g/ml)


    A-6427
    Vma 2
    V-ATP酶60kDa亚基
    真空膜


    A-6429
    Dpm 1
    Dol-P-Man合成
    ER


    A-6457
    PGK
    3-磷酸甘油酯
    细胞质




    激酶




食谱

  1. 球形缓冲液
    球形缓冲液
    100 ml
    200 ml
    1 M山梨醇(FW 182.17)


    10mM NaN 3 sub

    10μg/μl溶血酶或20〜40U/OD溶血酶


    40mM HEPES-KOH(pH7.5)


  2. 裂解缓冲液
    裂解缓冲液
    100 ml
    200 ml
    0.2M山梨醇(FW 182.17)


    50mM KOAc


    2mM EDTA

    20mM HEPES-KOH(pH 6.8)



  3. 将蛋白酶抑制剂加至终浓度(20μg/ml PMSF,5μg/ml抗蛋白酶,1μg/ml抑肽酶,亮抑蛋白酶肽和胃蛋白酶抑制剂,以及10μg/mlα亚基) - 球蛋白)。

致谢

该协议已经在约翰霍普金斯医学院的Espenshade实验室中修改和改编。 资助支持不同的项目,使用这个协议来自NIH - 国家心脏,肺和血液研究所,国家过敏和传染病研究所,胰腺癌行动网络和美国心脏协会。

参考

  1. Cowles,C.R.,Odorizzi,G.,Payne,G.S。和Emr,S.D。(1997)。 AP-3衔接子复合物对于货物选择性转运至酵母液泡至关重要。 Cell 91(1):109-118。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Tong, Z. (2012). Subcellular Fractionation Using Accudenz Gradient to Separate ER/Golgi in Yeast. Bio-protocol 2(2): e20. DOI: 10.21769/BioProtoc.20.
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