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Radioactive Pulse-Chase Analysis and Immunoprecipitation
放射性脉冲追踪分析和免疫沉淀法   

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Abstract

Labeling of newly-synthesized polypeptides with radioactive amino acids followed by immunoprecipitation allows quantitative analysis of the fate of a given protein in a time-dependent manner. This biochemical approach is usually used to study a variety of processes, such as protein folding, co-translational modifications, intracellular transport, and even its rate of degradation. Here, I describe step by step a simple technique to both label newly-synthesized influenza A virus (IAV) hemagglutinin (HA) with [35S]-methionine and then follow its maturation and transport through the secretory pathway by SDS-PAGE and fluorography (Magadan et al., 2013).

Materials and Reagents

  1. ~5 x 106 MDCK cells (ATCC, catalog number: CCL-34 )
  2. 0.05% 1x Trypsin-EDTA (phenol red) (Life Technologies, catalog number: 25300054 )
  3. DPBS (Life Technologies, catalog number: 14190-250 )
  4. Anti-HA antibodies (mostly home-made)
  5. nProtein A Sepharose 4 Fast Flow (GE, catalog number: 17-5280-02 )
  6. 4x NuPAGE LDS sample buffer (Life Technologies, catalog number: NP0007 )
  7. NuPAGE Novex 4-12% Bis-Tris protein gels (Life Technologies, catalog number: NP0321PK2 )
  8. NuPAGE MES SDS running buffer (Life Technologies, catalog number: NP000202 )
  9. DMEM media without methionine and cysteine (Life Technologies, catalog number: 21013024 )
  10. EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693159001 )
  11. 10% Triton X-100 surfact-amps detergent solution (Thermo Fisher Scientific, catalog number: 28314 )
  12. UltraPure 1 M Tris-HCI Buffer (pH 7.5) (Life Technologies, catalog number: 15567-027 )
  13. UltraPure 5 M NaCl (Life Technologies, catalog number: 24740-011 )
  14. UltraPure 0.5 M EDTA (pH 8.0) (Life Technologies, catalog number: 15575-020 )
  15. Methanol (Sigma-Aldrich, catalog number: 322415 )
  16. Acetic acid (Sigma-Aldrich, catalog number: 320099 )
  17. DMEM media (Life Technologies, catalog number: 10569-010 )
  18. DMEM media with 7.5% fetal bovine serum (see Recipes)
  19. Pulse medium (see Recipes)
  20. Chase medium (see Recipes)
  21. Non-denaturing lysis buffer (see Recipes)
  22. Fixation solution (see Recipes)
  23. L-cysteine (Sigma-Aldrich, catalog number: W326305 ) (see Recipes)
  24. [35S]-methionine (PerkinElmer, catalog number: NEG-709-A005MC ) (see Recipes)
  25. L-methionine (Sigma-Aldrich, catalog number: M9625 ) (see Recipes)

Equipment

Note: Designated for working with radioactive materials.

  1. Appropriate containers to dispose liquid and solid [35S]-waste following local radiation safety guidelines
  2. T-75 tissue culture flask (Thermo Fisher Scientific)
  3. 50 ml BD Falcon tube (BD Biosciences, Falcon®)
  4. A water bath set at 37 °C
  5. A refrigerated micro-centrifuge
  6. A rotator for 1.5 ml micro-centrifuge tubes settled at 4 °C
  7. 1.5 ml micro-centrifuge tubes
  8. A chamber to run mini-gels [I routinely use the XCell SureLock Mini Cell electrophoresis system (Life Technologies, catalog number: EI0001 ).]
  9. Carestream Kodak BioMax MR films (Sigma-Aldrich, catalog number: Z350400 )
  10. Kodak X-OMAT 2000A processor
  11. A gel-dryer
  12. A cassette for autoradiography

Procedure

  1. Radioactive Pulse-Chase
    1. Detach MDCK cells [grown in a T-75 tissue culture flask and infected with 10 infectious doses of influenza A/Puerto Rico/8/34 (PR8) virus per cell for 5 h at 37 °C (Magadan et al., 2013)] by incubating them with 5 ml Trypsin-EDTA for ~15 min at 37 °C.
    2. Transfer cells to a 50 ml BD Falcon tube and wash them twice with 10 ml pre-warmed DPBS by centrifugation for 1 min at 2,500 x g, room temperature.
    3. Resuspend cells in 1 ml DPBS and transfer them to a 1.5 ml micro-centrifuge tube.
    4. Centrifuge cells for 15 sec at max speed, 4 °C.
    5. Resuspend cells in 200 µl of pre-warmed pulse medium.
    6. Incubate cells for 2 min at 37 °C in a water bath (pulse).
    7. Centrifuge cells for 15 sec at max speed, 4 °C.
    8. Aspirate the supernatant and resuspend the cell pellet in 1.05 ml of pre-warmed chase medium.
    9. Incubate labeled cells for up to 20 min at 37 °C in a water bath.
    10. Take aliquots of 190 µl each at 0, 5, 10, 15, and 20 min and immediately transfer them to new 1.5 ml micro-centrifuge tubes containing 1 ml of ice-cold DPBS.
    11. Centrifuge cells for 15 sec at max speed, 4 °C.
    12. Aspirate the supernatant and resuspend the cell pellet in 1 ml ice-cold non-denaturing lysis buffer.
    13. Incubate cell lysates for 30 min at 4 °C with slow rotation.
    14. Clear cell lysates by centrifugation for 15 min at max speed, 4 °C.
    15. Keep the supernatant but discard the pellet.

  2. Immunoprecipitation
    1. Wash 30 µl of protein A sepharose/each chase-time point twice with 0.5 ml of ice-cold DPBS by centrifugation for 1 min at 3,000 x g, 4 °C.
    2. Resuspend resin in 0.5 ml of ice-cold DPBS supplemented with 0.001% Triton X-100 and the anti-HA antibody of choice.
    3. Incubate for at least 2 h at 4 °C with slow rotation.
    4. Wash resin twice with 0.5 ml of ice-cold non-denaturing lysis buffer (without protease inhibitors).
    5. Add 10 µl 10% BSA.
    6. Add the cleared cell lysate from each chase-time point.
    7. Incubate for at least 2 h at 4 °C with slow rotation.
    8. Wash resin twice with 0.5 ml of ice-cold non-denaturing lysis buffer (without protease inhibitors) but containing 0.1% instead of 0.5% Triton X-100.
    9. Wash resin once with 0.5 ml of ice-cold DPBS.
    10. Resuspend resin in 20 µl of 4x LDS sample buffer.
    11. Boil samples for 5 min.

  3. SDS-PAGE and Fluorography
    1. Load 15 µl of every sample onto protein mini-gels.
    2. Run for ~3 h at constant 50 mA/gel.
    3. Fix gels with 10 ml fixation solution for at least 30 min at room temperature with slow rocking.
    4. Dry gels in a gel drier for 1.5 h at 80 °C (in the case of 1 mm gels).
    5. Expose films to the radioactive gels overnight at room temperature.
    6. I usually develop my films using a Kodak X-OMAT 2000A processor.
    7. Please refer to Figure 1 on our prior publication (Magadan et al., 2013) for representative results and conclusions.

Recipes

  1. DMEM media with 7.5% fetal bovine serum
    Supplement DMEM media with 7.5% fetal bovine serum by adding ~40 ml commercial stock to 500 ml liquid media
  2. Pulse medium
    DMEM media without methionine and cysteine supplemented with 0.20 mM L-cysteine and 0.2 mCi/ml [35S]-methionine
  3. Chase medium
    DMEM media supplemented with 7.5% fetal bovine serum and 67 mM L-methionine
  4. Non-denaturing lysis buffer
    0.5% Triton X-100
    50 mM Tris-HCl (pH 7.5)
    300 mM NaCl
    5 mM EDTA
    Complete mini, EDTA-free protease inhibitor cocktail
  5. Fixation solution
    50% methanol
    10% acetic acid
  6. L-cysteine
    Dissolve 12.116 mg commercial powder in 500 ml DMEM media without methionine and cysteine to obtain a new media supplemented with 0.20 mM L-cysteine
  7. [35S]-methionine
    Add 19.6 µl of the 5 mCi/0.49 ml stock solution to 1 ml DMEM media without methionine to get a 0.2 mCi/ml working solution
  8. L-methionine
    Dissolve 10 mg commercial powder in 1 ml DMEM media + 7.5% fetal bovine serum to obtain a chase media supplemented with 67 mM L-methionine

Acknowledgments

This protocol has been adapted from a previously published paper (Magadan et al., 2013). This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

References

  1. Magadan, J. G., Khurana, S., Das, S. R., Frank, G. M., Stevens, J., Golding, H., Bennink, J. R. and Yewdell, J. W. (2013). Influenza A virus hemagglutinin trimerization completes monomer folding and antigenicity. J Virol 87(17): 9742-9753.

简介

用放射性氨基酸标记新合成的多肽,随后免疫沉淀允许以时间依赖性方式定量分析给定蛋白质的命运。 这种生物化学方法通常用于研究各种过程,如蛋白质折叠,共翻译修饰,细胞内转运,甚至其降解速率。 在这里,我逐步描述一个简单的技术,以标记新合成的甲型流感病毒(IAV)血凝素(HA)与[35 S] - 甲硫氨酸,然后跟随其成熟和运输通过分泌 通过SDS-PAGE和荧光成像(Magadan等人,2013)。

材料和试剂

  1. 〜5×10 6个MDCK细胞(ATCC,目录号:CCL-34)
  2. 0.05%1×胰蛋白酶-EDTA(酚红)(Life Technologies,目录号:25300054)
  3. DPBS(Life Technologies,目录号:14190-250)
  4. 抗HA抗体(大部分是自制的)
  5. nProtein A Sepharose 4 Fast Flow(GE,目录号:17-5280-02)
  6. 4x NuPAGE LDS样品缓冲液(Life Technologies,目录号:NP0007)
  7. NuPAGE Novex 4-12%Bis-Tris蛋白凝胶(Life Technologies,目录号:NP0321PK2)
  8. NuPAGE MES SDS运行缓冲液(Life Technologies,目录号:NP000202)
  9. 没有甲硫氨酸和半胱氨酸的DMEM培养基(Life Technologies,目录号:21013024)
  10. 无EDTA蛋白酶抑制剂混合物(Roche Diagnostics,目录号:04693159001)
  11. 10%Triton X-100表面活性剂洗涤剂溶液(Thermo Fisher Scientific,目录号:28314)
  12. UltraPure 1M Tris-HCl缓冲液(pH7.5)(Life Technologies,目录号:15567-027)
  13. UltraPure 5M NaCl(Life Technologies,目录号:24740-011)
  14. UltraPure 0.5M EDTA(pH 8.0)(Life Technologies,目录号:15575-020)
  15. 甲醇(Sigma-Aldrich,目录号:322415)
  16. 乙酸(Sigma-Aldrich,目录号:320099)
  17. DMEM培养基(Life Technologies,目录号:10569-010)
  18. 具有7.5%胎牛血清的DMEM培养基(参见Recipes)
  19. 脉冲介质(见配方)
  20. Chase介质(见配方)
  21. 非变性裂解缓冲液(参见配方)
  22. 固定解决方案(参见配方)
  23. L-半胱氨酸(Sigma-Aldrich,目录号:W326305)(参见Recipes)
  24. [PerkinElmer,目录号:NEG-709-A005MC](参见配方)。
  25. L-甲硫氨酸(Sigma-Aldrich,目录号:M9625)(参见Recipes)

设备

注意:指定用于使用放射性材料。

  1. 按照当地辐射安全指南处理液体和固体的适当容器[ 35 S] -
  2. T-75组织培养瓶(Thermo Fisher Scientific)
  3. 50ml BD Falcon管(BD Biosciences,Falcon )
  4. 设置在37℃的水浴
  5. 冷冻微型离心机
  6. 用于1.5ml微量离心管的旋转器在4℃下固定
  7. 1.5 ml微量离心管
  8. 运行微型凝胶的小室[我常规使用XCell SureLock Mini Cell电泳系统(Life Technologies,目录号:EI0001)。]
  9. Carestream Kodak BioMax MR膜(Sigma-Aldrich,目录号:Z350400)
  10. 柯达X-OMAT 2000A处理器
  11. 凝胶干燥器
  12. 用于放射自显影的暗盒

程序

  1. 放射性脉冲追踪
    1. 分离MDCK细胞[在T-75组织培养瓶中生长,并在37℃下用10个感染剂量的流感A /波多黎各/8/34(PR8)病毒每个细胞感染5小时(Magadan等, ,2013)] 在37℃下用5ml胰蛋白酶-EDTA孵育约15分钟
    2. 转移细胞到50ml BD Falcon管,并用10ml预热的DPBS通过在室温下以2,500×g离心1分钟洗涤它们两次。
    3. 重悬细胞在1 ml DPBS,并将其转移到1.5毫升微量离心管。
    4. 离心细胞15秒,最高速度,4℃
    5. 重悬细胞在200微升的预热脉冲介质
    6. 在37℃水浴(脉冲)孵育细胞2分钟
    7. 离心细胞15秒,最高速度,4℃
    8. 吸出上清液,并在1.05ml预热的追踪培养基中重悬细胞沉淀
    9. 在37℃下,在水浴中孵育标记的细胞达20分钟
    10. 在0,5,10,15和20分钟取每份190μl的等分试样,立即将其转移到含有1ml冰冷的DPBS的新的1.5ml微量离心管中。
    11. 离心细胞15秒,最高速度,4℃
    12. 吸出上清液,并在1ml冰冷的非变性裂解缓冲液中重悬细胞沉淀
    13. 孵育细胞裂解液30分钟,在4°C,慢旋转
    14. 通过在4℃下以最大速度离心15分钟来澄清细胞裂解物
    15. 保持上清液,但丢弃沉淀。

  2. 免疫沉淀
    1. 通过在3,000xg,4℃下离心1分钟,用0.5ml冰冷的DPBS洗涤30μl蛋白A琼脂糖/每个追踪时间点两次。
    2. 将树脂重悬于补充有0.001%Triton X-100和所选抗HA抗体的0.5ml冰冷的DPBS中。
    3. 在4℃下缓慢旋转孵育至少2小时。
    4. 用0.5ml冰冷的非变性裂解缓冲液(无蛋白酶抑制剂)洗涤树脂两次
    5. 加入10μl10%BSA
    6. 添加从每个追逐时间点清除的细胞裂解物。
    7. 在4℃下缓慢旋转孵育至少2小时。
    8. 用0.5ml冰冷的非变性裂解缓冲液(无蛋白酶抑制剂)洗涤树脂两次,但含有0.1%而不是0.5%Triton X-100。
    9. 用0.5ml冰冷的DPBS洗涤树脂一次。
    10. 将树脂重悬在20μl的4x LDS样品缓冲液中
    11. 煮沸样品5分钟。

  3. SDS-PAGE和荧光成像
    1. 加载15微升的每个样品到蛋白质小凝胶上
    2. 在恒定的50mA /凝胶下运行〜3小时
    3. 用10ml固定溶液在室温下用慢摇动固定凝胶至少30分钟
    4. 在凝胶干燥器中在80℃下干凝胶1.5小时(在1mm凝胶的情况下)
    5. 在室温下将膜暴露于放射性凝胶过夜。
    6. 我通常使用Kodak X-OMAT 2000A处理器开发我的电影
    7. 请参阅我们之前的出版物(Magadan等人,2013年)的图1,了解具有代表性的结果和结论。

食谱

  1. 含7.5%胎牛血清的DMEM培养基 补充含7.5%胎牛血清的DMEM培养基,通过向500ml液体培养基中加入约40ml商业原液
  2. 脉冲介质
    没有添加0.20mM L-半胱氨酸和0.2mCi/ml [35 S] - 蛋氨酸的甲硫氨酸和半胱氨酸的DMEM培养基
  3. 追逐媒体
    补充有7.5%胎牛血清和67mM L-甲硫氨酸的DMEM培养基
  4. 非变性裂解缓冲液
    0.5%Triton X-100 50mM Tris-HCl(pH7.5) 300 mM NaCl
    5 mM EDTA
    完全微型,无EDTA蛋白酶抑制剂混合物
  5. 固定溶液
    50%甲醇
    10%乙酸
  6. L-半胱氨酸
    将12.116mg商业粉末溶解在不含甲硫氨酸和半胱氨酸的500ml DMEM培养基中,得到补充有0.20mM L-半胱氨酸的新培养基。
  7. [s] 35 S] - 甲硫氨酸 将19.6μl5 mCi/0.49 ml储备溶液加入1 ml不含蛋氨酸的DMEM培养基中,得到0.2 mCi/ml工作溶液。
  8. L-甲硫氨酸 将10mg商品粉末溶解在1ml DMEM培养基+ 7.5%胎牛血清中,得到补充有67mM L-甲硫氨酸的追加培养基

致谢

该协议已经改编自以前发表的论文(Magadan等人,2013年)。 这项工作是由国家研究所的过敏和传染病,国家卫生研究院的校内研究支持。

参考文献

  1. Magadan,J.G.,Khurana,S.,Das,S.R.,Frank,G.M.,Stevens,J.,Golding,H.,Bennink,J.R.and Yewdell,J.W。(2013)。 甲型流感病毒血凝素三聚化完成单体折叠和抗原性。/em> 87(17):9742-9753。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Magadán, J. G. (2014). Radioactive Pulse-Chase Analysis and Immunoprecipitation. Bio-protocol 4(8): e1101. DOI: 10.21769/BioProtoc.1101.
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