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Analysis of Protein Stability by the Cycloheximide Chase Assay
放线菌酮追踪检测分析蛋白质的稳定性   

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参见作者原研究论文

本实验方案简略版
Oncogene
Jun 2014

Abstract

Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.

Materials and Reagents

  1. Cell lines
    1. CL1-5/Flag-wtSlug stable cells
    2. CL1-5/Flag-4SA stable cells

  2. Antibodies
    1. Flag M2 antibodies (Sigma-Aldrich, catalog number: F3165 )
    2. β–actin antibodies (Sigma-Aldrich, catalog number: A5441 )

  3. Buffer and media
    1. 100 mg/ml cycloheximide (CHX) stock solution (Sigma-Aldrich, catalog number: C7698 )
    2. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-176 )
    3. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10082147 )
    4. HEPES (Sigma-Aldrich, catalog number: H3784 )
    5. Streptomycin/Penicillin (5,000 U/ml) (Life Technologies, Gibco®, catalog number: 15070063 )
    6. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 )
    7. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
    8. G418 (Sigma-Aldrich, catalog number: A1720 )
    9. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
    10. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
    11. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
    12. Sodium fluoride (Sigma-Aldrich, catalog number: S7920 )
    13. Sodium pyrophosphate (Sigma-Aldrich, catalog number: 221368 )
    14. Protease inhibitor cocktail with EDTA (Roche, Catalog number: 04693116001 )
    15. BCA protein assay kit (Pierce, catalog number: 23225 )
    16. DTT (Sigma-Aldrich, catalog number: 43815 )
    17. SDS (Sigma-Aldrich, catalog number: L3771 )
    18. Glycerol (Sigma-Aldrich, catalog number: G5516 )
    19. Bromophenoblue (Sigma-Aldrich, catalog number: B5525 )
    20. Complete medium (see Recipes)
    21. Protein lysis buffer (see Recipes)
    22. 5x sample buffer (see Recipes)

Equipment

  1. CO2 incubator (Thermo Fisher Scientific, Forma series II)
  2. 35-mm culture dishes (Corning, catalog number: 430165 )
  3. 1.5 ml tubes
  4. Standard sonicator

Software

  1. MetaMorph software (Molecular Devices)

Procedure

Determine the time course of cycloheximide chase beforehand. There can be great variation among proteins. For an unfamiliar protein, it is recommended to start with a 4-hour interval and chase till 24 h. Cycloheximide may cause cytotoxicity to certain cells if cells are exposed to it over 20-24 h. It has been known that Slug is a labile protein with a T1/2 of about 40 min (Wang et al., 2009). CL1-5 cells stably overexpressing Flag-tagged wtSlug and non-phosphorylatable Slug-4SA were prepared using lentiviral transduction (Kao et al., 2014). Cells were grown in complete medium with 400 μg/ml G418. To compare the turnover of wtSlug and Slug-4SA, these stable cells were subjected to the cycloheximide chase assay.

  1. Seed about 6 x 105 cells in 35-mm dishes and have them incubated in a CO2 incubator overnight.
  2. After 12-h incubation, remove the medium and add complete medium with 300 μg/ml* cycloheximide (dissolved in DMSO) into each dish.
    *The concentration of cycloheximide depends on the cell line used. For CL1-5 cells, 300 μg/ml cyclcoheximide does not cause apoptosis within the time course of the chase assay. It is recommended to start a cell line test with different concentrations of cycloheximide (ranging from 50-300 μg/ml) for a least 8-h chase. For transfected cells, to avoid the sub-efficient cycloheximide concentrations, the expression of tagged proteins should be determined in the absence or presence of different concentrations of cycloheximide by Western blot at 24 h after transfection.
  3. Prepare the protein lysis buffer with freshly-added protease inhibitor. About 200 μl of lysis buffer is needed for one 35-mm dish.
  4. Lyse t = 0 h cells with the protein lysis buffer and store the lysates in a -80 °C freezer.
  5. Collect lysates at other time points (t = 1, 2, 3, 6 h) according to the experimental design.
  6. After all the lysates have been collected, sonicate the protein lysates with a sonicator for 10 times (1 sec/time) on ice.
  7. Centrifuge lysates at 12,000 rpm at 4 °C for 30 min.
  8. Collect and transfer the supernatants to new tubes with corresponding labels.
  9. Determine protein concentrations by the BCA protein assay kit.
  10. Take 50 μg of proteins, add 5x sample buffer, and incubate samples at 100 °C for 10 min.
  11. Analyze the results by the SDS-PAGE assay and Western blotting (anti-Flag M2 antibodies with 1:5,000 dilution; β-actin antibodies with 1:10,000 dilution).
  12. Quantify the Western bands of Flag and β-actin in triplicates using MetaMorph Software or other equivalent softwares.

Quantification

Quantification was achieved by the MetaMorph software in this case. An analysis guide should be referred to for a first-time user. Basically, regions of measurement are selected and threshold is defined. Draw region of interest on top of the western blot bands and quantitate its total grey signal (this can be done in Image J and any other image analysis software). The values of total thresholded areas are measured; a ratio of Flag-Slug to its β-actin is then calculated. The final Slug protein turnover rate at each time point is the percentage of Slug/β-actin at t = 0 of each experimental group. A plot can be created afterwards.

Representative data

Western blots and quantification images have been published in Oncogene (Kao et al., 2014). For details, please refer to Figure 2f at Kao et al. (2014).

Recipes

  1. Complete medium
    RPMI-1640
    10% FBS
    15 mM HEPES (pH 7.01)
    100 U/ml streptomycin/penicillin
    1 mM sodium pyruvate
    44 mM sodium bicarbonate
  2. Protein lysis buffer
    20 mM Tris (pH 7.5)
    100 mM NaCl
    1% IGEPAL CA-630
    100 mM Na3VO4
    50 mM NaF
    30 mM sodium pyrophosphate
  3. 5x sample buffer
    250 mM Tris-HCl (pH 6.8)
    500 mM DTT
    10% SDS
    50% glycerol
    0.1% bromophenoblue

Acknowledgments

This work was supported by grants from the National Science Council, Taiwan (NSC99-2628-B-006-031-MY3 NSC101-2325-B-006-018, NSC100-2321-B-002-071 and NSC101-2321-B002-068), National Taiwan University, Taiwan (10R71601-2), and National Institute of Health, USA (R01-GM-094231, to AIN). SP Wang is supported by a Human Frontier Science Program long-term fellowship. This protocol was adapted from previous work published in Oncogene (Kao et al., 2014).

References

  1. Kao, S. H., Wang, W. L., Chen, C. Y., Chang, Y. L., Wu, Y. Y., Wang, Y. T., Wang, S. P., Nesvizhskii, A. I., Chen, Y. J., Hong, T. M. and Yang, P. C. (2014). GSK3beta controls epithelial-mesenchymal transition and tumor metastasis by CHIP-mediated degradation of Slug. Oncogene 33(24): 3172-3182.
  2. Wang, S. P., Wang, W. L., Chang, Y. L., Wu, C. T., Chao, Y. C., Kao, S. H., Yuan, A., Lin, C. W., Yang, S. C., Chan, W. K., Li, K. C., Hong, T. M. and Yang, P. C. (2009). p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug. Nat Cell Biol 11(6): 694-704.

简介

真核细胞中蛋白质稳定性的比较已经通过放线菌酮实现,放线菌酮是蛋白质生物合成的抑制剂,由于其在翻译延伸中的预防。 它在确定给定蛋白质的半衰期方面广泛用于细胞生物学,并且已在癌症研究中获得广泛应用。 在这里,我们在我们的实验室使用肺腺癌细胞系CL1-5作为模型呈现完整的放线菌酮追踪测定法。

材料和试剂

  1. 单元格行
    1. CL1-5/Flag-wtSlug稳定单元格
    2. CL1-5/Flag-4SA稳定细胞

  2. 抗体
    1. Flag M2抗体(Sigma-Aldrich,目录号:F3165)
    2. β-肌动蛋白抗体(Sigma-Aldrich,目录号:A5441)

  3. 缓冲区和媒体
    1. 100mg/ml环己酰亚胺(CHX)储备溶液(Sigma-Aldrich,目录号:C7698)
    2. RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875-176)
    3. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10082147)
    4. HEPES(Sigma-Aldrich,目录号:H3784)
    5. 链霉素/青霉素(5,000U/ml)(Life Technologies,Gibco ,目录号:15070063)
    6. 丙酮酸钠(Sigma-Aldrich,目录号:P5280)
    7. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
    8. G418(Sigma-Aldrich,目录号:A1720)
    9. 氯化钠(Sigma-Aldrich,目录号:S7653)
    10. IGEPAL CA-630(Sigma-Aldrich,目录号:I8896)
    11. 原钒酸钠(Sigma-Aldrich,目录号:S6508)
    12. 氟化钠(Sigma-Aldrich,目录号:S7920)
    13. 焦磷酸钠(Sigma-Aldrich,目录号:221368)
    14. 蛋白酶抑制剂混合物与EDTA(Roche,目录号:04693116001)
    15. BCA蛋白测定试剂盒(Pierce,目录号:23225)
    16. DTT(Sigma-Aldrich,目录号:43815)
    17. SDS(Sigma-Aldrich,目录号:L3771)
    18. 甘油(Sigma-Aldrich,目录号:G5516)
    19. 溴苯酚(Sigma-Aldrich,目录号:B5525)
    20. 完整介质(见配方)
    21. 蛋白裂解缓冲液(参见配方)
    22. 5x样品缓冲液(参见配方)

设备

  1. CO 2培养箱(Thermo Fisher Scientific,Forma系列II)
  2. 35-mm培养皿(Corning,目录号:430165)
  3. 1.5 ml管
  4. 标准超声仪

软件

  1. MetaMorph软件(Molecular Devices)

程序

事先确定放线菌酮追踪的时间过程。 蛋白质之间可能存在巨大差异。 对于不熟悉的蛋白质,建议以4小时的间隔开始,并追踪至24小时。 如果细胞暴露于20-24小时,环己酰亚胺可能对某些细胞产生细胞毒性。 已知Slug是具有约40分钟的T 1/2 1/2的不稳定蛋白质(Wang等人,2009)。 使用慢病毒转导制备稳定过表达Flag标记的wtSlug和不可磷酸化Slug-4SA的CL1-5细胞(Kao等人,2014)。 细胞在具有400μg/ml G418的完全培养基中生长。 为了比较wtSlug和Slug-4SA的转换,将这些稳定的细胞进行放线菌酮追踪测定。

  1. 在35-mm培养皿中接种约6×10 5个细胞,并将它们在CO 2培养箱中温育过夜。
  2. 在12小时温育后,除去培养基并向每个培养皿中加入含有300μg/ml放线菌酮(溶于DMSO)的完全培养基。
    *放线菌酮的浓度取决于所用的细胞系。对于CL1-5细胞,300μg/ml环己酰亚胺在追踪测定的时间过程中不引起凋亡。建议开始不同浓度的放线菌酮(范围从50-300μg/ml)的细胞系测试至少8小时追逐。对于转染的细胞,为了避免次有效的放线菌酮浓度,应在转染后24小时通过蛋白质印迹在不存在或存在不同浓度的放线菌酮时测定标记蛋白的表达。
  3. 用新添加的蛋白酶抑制剂制备蛋白裂解缓冲液。一个35-mm培养皿需要约200μl裂解缓冲液。
  4. 用蛋白裂解缓冲液裂解t = 0h细胞,并将裂解物储存在-80℃冰箱中。
  5. 根据实验设计,在其他时间点(t = 1,2,3,6h)收集溶胞产物
  6. 在收集所有裂解物后,用超声波仪在冰上超声处理蛋白裂解物10次(1秒/次)。
  7. 离心裂解液在12,000 rpm在4°C 30分钟。
  8. 收集上清液并将其转移到具有相应标记物的新管中。
  9. 通过BCA蛋白测定试剂盒确定蛋白浓度
  10. 取50μg蛋白质,加入5x样品缓冲液,并在100℃孵育样品10分钟
  11. 通过SDS-PAGE测定和Western印迹(具有1:5,000稀释的抗Flag M2抗体;具有1:10,000稀释的β-肌动蛋白抗体)分析结果。
  12. 使用MetaMorph软件或其他等效软件,一式三份量化Flag和β-actin的Western谱带。

定量

在这种情况下通过MetaMorph软件实现定量。第一次用户应参考分析指南。基本上,选择测量区域并定义阈值。在Western印迹带的顶部绘制感兴趣的区域并定量其总灰度信号(这可以在图像J和任何其他图像分析软件中进行)。测量总阈值面积的值;然后计算Flag-Slug与其β-肌动蛋白的比率。每个时间点的最终Slug蛋白转换率是每个实验组在t = 0时Slug /β-肌动蛋白的百分比。之后可以创建绘图。

代表数据

Western杂交和定量图像已经在Oncogene中发表(Kao等人,2014)。有关详细信息,请参阅图2f(Kao em et al。)。(2014)。

食谱

  1. 完成媒介
    RPMI-1640
    10%FBS
    15mM HEPES(pH 7.01)
    100 U/ml链霉素/青霉素
    1mM丙酮酸钠 44mM碳酸氢钠
  2. 蛋白裂解缓冲液
    20mM Tris(pH7.5) 100 mM NaCl
    1%IGEPAL CA-630
    100mM Na 3+ VO 4
    50mM NaF 食谱

    1. 完成媒介
      RPMI-1640
      10%FBS
      15mM HEPES(pH 7.01)
      100 U/ml链霉素/青霉素
      1mM丙酮酸钠 44mM碳酸氢钠
    2. 蛋白裂解缓冲液
      20mM Tris(pH7.5) 100 mM NaCl
      1%IGEPAL CA-630
      100mM Na 3+ VO 4
      50mM NaF... 食谱

      1. 完成媒介
        RPMI-1640
        10%FBS
        15mM HEPES(pH 7.01)
        100 U/ml链霉素/青霉素
        1mM丙酮酸钠 44mM碳酸氢钠
      2. 蛋白裂解缓冲液
        20mM Tris(pH7.5) 100 mM NaCl
        1%IGEPAL CA-630
        100mM Na 3+ VO 4
        50mM NaF......
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kao, S., Wang, W., Chen, C., Chang, Y., Wu, Y., Wang, Y., Wang, S., Nesvizhskii, A. I., Chen, Y., Hong, T. and Yang, P. (2015). Analysis of Protein Stability by the Cycloheximide Chase Assay. Bio-protocol 5(1): e1374. DOI: 10.21769/BioProtoc.1374.
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Tao Zhang
Institute of Neuroscience, CAS
Hello,I have two questions. Do I need to mixing the lysate adequately before transfer it to -80? How could I determine the concentration of CHX when I use the cells of rat embryonic fibroblast. Should I treated the cells with CHX with different concentrations for 8 hours and observe the apoptosis markers and some protein with well-known half-life? Thank you very much.
9/2/2015 1:08:52 AM Reply