搜索

Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells
从H1 HeLa细胞中分离放射性标记骨髓灰质炎病毒颗粒   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The following protocol describes the isolation of radioactive viral and subviral particles from infected cells. This protocol has been written for isolation of poliovirus particles from H1 HeLa cells. Infection protocol and timing of [35S] methionine labeling and particle collection should be tailored to the virus of interest. Following isolation of the viral particles, the viral proteins present in these particles may be separated by gel electrophoresis and visualized by autoradiography.

Materials and Reagents

  1. H1 HeLa cells
  2. Poliovirus
  3. Sucrose (Life Technologies, InvitrogenTM, catalog number: 15503-022 )
  4. Methionine free DMEM (Life Technologies, Gibco®, catalog number: 21013 )
  5. MEM (Life Technologies, Gibco®, catalog number: 11095-080 )
  6. [35S] Methionine (PerkinElmer, catalog number: NEG772002MC )
  7. Mineral oil (Sigma-Aldrich, catalog number: M5904 )
  8. SDS-PAGE gel
  9. Nonidet P-40 (NP-40) (Sigma-Aldrich, catalog number: N-6507 )
  10. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P3075-1G )
  11. 40 U RNAsin® Ribonuclease Inhibitor (Promega Corporation, catalog number: N2111 )
  12. 4x Laemelli loading buffer
  13. Tris buffer (see Recipes)
  14. Lysis buffer (see Recipes)
  15. Gel Fixative (see Recipes)
  16. Phosphate Buffered Saline (PBS) (see Recipes)
  17. PBS+ (see Recipes)

Equipment

  1. Centrifuge tubes (Beckman Coulter, catalog number: 344059 )
  2. 1.5 ml Eppendorf tubes
  3. Peristaltic Pump (Bio-Rad Laboratories, model: 731-9001EDU )
  4. Ultracentrifuge (Beckman Coulter, model: Optima L-90K )
  5. SW41 Ti rotor (Beckman Coulter, model: 331362 )
  6. Fraction collection system (Beckman Coulter, catalog number: 270-331580 )
  7. Gradient maker (Biocomp Gradient Master, model: 107 )
  8. Liquid scintillation counter (Beckman Coulter, model: LS6000IC )
  9. Gel dryer (Bio-Rad Laboratories, model: 583 )

Procedure

  1. Isolation of viral and subviral particles
    1. Infect adherent cells in 10 cm dish at a MOI of 50. The virus should be diluted in 500 μl of PBS+. Incubate cells with virus alone for 30 min at 37 °C prior to the addition of 8 ml MEM. Cells should be approximately 80% confluent at the time of infection.
    2. Prepare [35S] methionine-DMEM by adding 100 μCi [35S] methionine per milliliter of methionine free DMEM.
    3. At 3 h post-infection remove media from cells, add 3 ml methionine free DMEM to cells then remove. Repeat this process for two additional washes, then add 6 ml of the [35S] methionine DMEM prepared in step A-2 to cells.
    4. At desired collection time points, remove [35S] methionine DMEM from cells. Wash cells 3x with 3 ml PBS and add 1 ml of lysis buffer to the plate. Remove the cells from the plate by pipetting up and down several times. Then transfer the lysate (cells & lysis buffer) to a 1.5 ml tube.
    5. Remove nuclei from this lysate by centrifugation at 4,500 x g for 10 min at 4 °C (store lysate on ice until it is added to the gradient).
    6. Prepare 15% (w/v) and 30% (w/v) sucrose solutions using Tris buffer.
    7. Follow gradient maker instructions for pouring a 15-30% sucrose gradient.
    8. Apply 500 μl of lysate to the top of the gradient. Add approximately 200 μl of mineral oil over the radiolabeled lysate.
    9. Spin tube in ultracentrifuge for 3 h at 27,500 rpm, 15 °C. The ultracentrifuge should be programmed for slow acceleration and deceleration with no brake.
    10. Place tube in fraction collection system.
    11. Turn on pump, to begin pumping air on top of the gradient. Immediately puncture the tube to allow sucrose to flow out of the bottom of the centrifuge tube.
    12. Collect 0.5 ml fractions from the gradient into 1.5 ml tubes. The entire gradient should be collected. Fractions may be stored at -20 °C for up to one year.
    13. Determine radioactivity in fractions using a liquid scintillation counter.
  2. Visualization of labeled proteins in fractions
    1. Mix 15 μl of each fraction with 5 μl of 4x Laemelli loading buffer.
    2. Heat sample to 95 °C.
    3. Separate proteins by SDS-PAGE using a 12% SDS-acrylamide gel.
    4. Fix gel overnight in gel fixative at room temperature with shaking.
    5. Dry gel using gel dryer.
    6. Visualize proteins using standard autoradiography techniques.

Recipes

  1. Tris buffer
    10 mM Tris (pH 7.4)
    10 mM NaCl
    1.5 mM MgCl2
  2. Lysis buffer
    10 ml Tris Buffer
    1% NP-40
    1 μM PMSF
    40 U RNAsin
  3. Gel Fixative
    50% methanol
    10% acetic acid in distilled H2O
  4. PBS (makes 1 L)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    Dissolve reagents in 800 ml ddH2O, adjust pH to 7.5, and then add ddH2O to 1 L.
    To prepare PBS+ add 1 mM CaCl2.2H2O and 0.5 mM MgCl2.6H2O.

Acknowledgments

This protocol is adapted from Richards and Jackson (2012).

References

  1. Richards, A. L. and Jackson, W. T. (2012). Intracellular vesicle acidification promotes maturation of infectious poliovirus particles. PLoS Pathog 8(11): e1003046.

简介

以下方案描述了从感染的细胞中分离放射性病毒和亚病毒颗粒。 该协议是为了从H1 HeLa细胞分离脊髓灰质炎病毒颗粒而编写的。 [35]甲硫氨酸标记和颗粒收集的感染方案和时机应该针对感兴趣的病毒定制。 分离病毒颗粒后,可以通过凝胶电泳分离存在于这些颗粒中的病毒蛋白,并通过放射自显影进行显现。

材料和试剂

  1. H1 HeLa细胞
  2. 脊髓灰质炎病毒
  3. 蔗糖(Life Technologies,Invitrogen TM,目录号:15503-022)
  4. 不含甲硫氨酸的DMEM(Life Technologies,Gibco ,目录号:21013)
  5. MEM(Life Technologies,Gibco ,目录号:11095-080)
  6. 甲硫氨酸(PerkinElmer,目录号:NEG772002MC)
  7. 矿物油(Sigma-Aldrich,目录号:M5904)
  8. SDS-PAGE凝胶
  9. Nonidet P-40(NP-40)(Sigma-Aldrich,目录号:N-6507)
  10. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P3075-1G)
  11. 40 U核糖核酸酶抑制剂(Promega Corporation,目录号:N2111)
  12. 4x Laemelli加载缓冲区
  13. Tris缓冲液(见配方)
  14. 裂解缓冲液(见配方)
  15. 凝胶固定剂(参见配方)
  16. 磷酸盐缓冲盐水(PBS)(见配方)
  17. PBS +(请参阅配方)

设备

  1. 离心管(Beckman Coulter,目录号:344059)
  2. 1.5 ml Eppendorf管
  3. 蠕动泵(Bio-Rad Laboratories,型号:731-9001EDU)
  4. 超速离心机(Beckman Coulter,型号:Optima L-90K)
  5. SW41Ti转子(Beckman Coulter,型号:331362)
  6. 馏分收集系统(Beckman Coulter,目录号:270-331580)
  7. 梯度仪(Biocomp Gradient Master,型号:107)
  8. 液体闪烁计数器(Beckman Coulter,型号:LS6000IC)
  9. 凝胶干燥器(Bio-Rad Laboratories,型号:583)

程序

  1. 病毒和亚病毒颗粒的分离
    1. 感染贴壁细胞在10厘米的菜,在MOI为50.该病毒应该稀释在500微升的PBS +。孵育细胞与病毒单独在37℃下30分钟,然后加入8毫升MEM。在感染时细胞应该约80%汇合
    2. 通过每毫升不含甲硫氨酸的DMEM加入100μCi[sup] 3S]甲硫氨酸制备[supra] S [甲硫氨酸-DMEM]。
    3. 在感染后3小时从细胞中除去培养基,向细胞中加入3ml不含甲硫氨酸的DMEM,然后除去。重复该过程两次另外的洗涤,然后将6ml步骤A-2中制备的[35 S]甲硫氨酸DMEM加入细胞。
    4. 在所需的收集时间点,从细胞中除去[sup] 3S]甲硫氨酸DMEM。用3ml PBS洗涤细胞3次,并向板中加入1ml裂解缓冲液。通过上下吹吸数次从板中取出细胞。然后将裂解物(细胞和裂解缓冲液)转移到1.5ml试管中
    5. 通过在4℃下以4,500×g离心10分钟(在冰上储存裂解物,直到其加入到梯度中),从该裂解物中移除核。
    6. 使用Tris缓冲液制备15%(w/v)和30%(w/v)蔗糖溶液
    7. 按照梯度制造商的说明,倾倒15-30%蔗糖梯度
    8. 应用500μl裂解物到梯度的顶部。 在放射性标记的裂解物上加入约200μl矿物油
    9. 在超速离心机中以27,500rpm,15℃离心3小时。 超速离心机应编程为无制动的慢加速和减速。
    10. 将管置于馏分收集系统中。
    11. 打开泵,开始在梯度顶部抽空空气。 立即刺破试管,使蔗糖流出离心管的底部。
    12. 从梯度中收集0.5ml级分到1.5ml管中。 应收集整个梯度。 级分可以在-20℃储存长达一年。
    13. 使用液体闪烁计数器确定级分中的放射性
  2. 标记蛋白质在级分中的可视化
    1. 混合15微升的每个部分与5微升的4x Laemelli加载缓冲液
    2. 将样品加热至95°C
    3. 使用12%SDS-丙烯酰胺凝胶通过SDS-PAGE分离蛋白质
    4. 在凝胶固定剂中在室温下摇动固定凝胶过夜。
    5. 使用凝胶干燥器的干凝胶
    6. 使用标准放射自显影技术可视化蛋白质。

食谱

  1. Tris缓冲液
    10mM Tris(pH7.4) 10mM NaCl 1.5mM MgCl 2·h/v
  2. 裂解缓冲液
    10ml Tris缓冲液
    1%NP-40
    1μMPMSF
    40 U RNAsin
  3. 凝胶固定剂
    50%甲醇
    10%乙酸的蒸馏水溶液中
  4. PBS(使1 L)
    137 mM NaCl 2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH 2 PO 4 sub/
    将试剂溶解在800ml ddH 2 O中,将pH调节至7.5,然后将ddH 2 O加至1L。
    为了制备PBS +,加入1mM CaCl 2 2,2H 2 O 2和0.5mM MgCl 2。/sup> 6H 2 O。

致谢

该协议改编自Richards和Jackson(2012)。

参考文献

  1. Richards,A.L.和Jackson,W.T。(2012)。 胞内囊泡酸化促进感染性脊髓灰质炎病毒颗粒的成熟。 PLoS Pathog 8(11):e1003046。
  • English
  • 中文翻译
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. Richards, A. and Jackson, W. (2013). Isolation of Radiolabeled Poliovirus Particles from H1 HeLa Cells. Bio-protocol 3(16): e872. DOI: 10.21769/BioProtoc.872.
  2. Richards, A. L. and Jackson, W. T. (2012). Intracellular vesicle acidification promotes maturation of infectious poliovirus particles. PLoS Pathog 8(11): e1003046.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。