放线菌酮追踪检测分析蛋白质的稳定性

Shih-Han Kao; Wen-Lung Wang; Chi-Yuan Chen; Yih-Leong Chang; Yi-Ying Wu; Yi-Ting Wang; Shu-Ping Wang; Alexey I Nesvizhskii; Yu-Ju Chen; Tse-Ming Hong; Pan-Chyr Yang

doi:10.21769/BioProtoc.1374

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Peer-reviewed

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

AN Alexey I Nesvizhskii

YC Yu-Ju Chen

TH Tse-Ming Hong

Pan-Chyr Yang email

发布: 2015年01月05日第5卷第1期 DOI: 10.21769/BioProtoc.1374 浏览次数: 62054

评审: Hsin-Yi ChangGuillermo GomezAnonymous reviewer(s)

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Cover of Oncogene, featuring study using the protocol.

Jun 2014

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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

Cell lines
1. CL1-5/Flag-wtSlug stable cells
2. CL1-5/Flag-4SA stable cells
Antibodies
1. Flag M2 antibodies (Sigma-Aldrich, catalog number: F3165 )
2. β–actin antibodies (Sigma-Aldrich, catalog number: A5441 )
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

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

Software

MetaMorph software (Molecular Devices)

Procedure

English

中文翻译

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如何引用

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.