Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Trypsin Sensitivity Assay to Study the Folding Status of Proteins

胰蛋白酶敏感性试验研究蛋白质的折叠状态

Satoshi Ninagawa Satoshi Ninagawa
Kazutoshi Mori Kazutoshi Mori

发布: 2016年10月05日第6卷第19期 DOI: 10.21769/BioProtoc.1953 浏览次数: 11298

评审: Ralph BottcherMelike ÇağlayanGregory C. Finnigan

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Cell Biology, featuring study using the protocol.

Nov 2015

Advertisement

“科研可重复性与创新”系列网络研讨会
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

采用模块化标签系统与连接型 mini-Gq 纯化活化状态 G 蛋白偶联受体 ADGRL4 以用于冷冻电镜研究

采用模块化标签系统与连接型 mini-Gq 纯化活化状态 G 蛋白偶联受体 ADGRL4 以用于冷冻电镜研究

David M. Favara and Christopher G. Tate

2026年03月05日 635 阅读

有丝分裂细胞中变性条件下的SUMO免疫沉淀方法

有丝分裂细胞中变性条件下的SUMO免疫沉淀方法

Alexandra K. Walker [...] Joanna R. Morris

2026年04月05日 382 阅读

利用有限蛋白水解质谱鉴定脑组织中的蛋白质结构差异

利用有限蛋白水解质谱鉴定脑组织中的蛋白质结构差异

Haley E. Tarbox and Stephen D. Fried

2026年05月05日 650 阅读

Abstract

This protocol aims to evaluate folding status of proteins, utilizing trypsin sensitivity. Unfolded/misfolded proteins are more susceptible to trypsin than folded proteins, because trypsin easily accesses and cleaves loosely folded parts of proteins. This method is especially useful to compare tightness of the folding among wild-type and mutant proteins. As trypsin generally cleaves a peptide bond at the carboxyl-terminal side of the amino acids lysine or arginine, this method can be used to analyze the folding status of different types of proteins such as integral membrane or soluble proteins (Ninagawa et al., 2015) and is applicable to cell lysates of any species and tissues as well as to recombinant proteins. You can use this technique with regular molecular and cell biology equipment.

Keywords: Trypsin (胰蛋白酶) search Protein folding (蛋白质折叠) search Integral membrane and soluble proteins (积分膜蛋白和可溶性) search

Materials and Reagents

  1. 6 well dish (Corning, Falcon®, catalog number: 353046 )
  2. PVDF membrane (GE Healthcare, catalog number: 10600023 )
  3. DT40 cell line (DT40 is a B cell line derived from an avian leukosis virus induced bursal lymphoma in a white leghorn chicken) (ATCC, catalog number: CRL-2111 )
  4. Homo sapiens colon colorectal carcinoma cell line (HCT116) (ATCC, catalog number: CCL-247 )
  5. Dulbecco’s modified Eagle’s medium (DMEM) (NACALAI TESQUE, catalog number: 08458-45 )
  6. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  7. 100 U/ml penicillin and 100 μg/ml streptomycin (NACALAI TESQUE)
  8. RPMI (NACALAI TESQUE, catalog number: 30263-95 )
  9. Chicken serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16110-082 )
  10. Opti-mem (Thermo Fisher Scientific, GibcoTM, catalog number: 31985-070 )
  11. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  12. 2.5 g/L trypsin (NACALAI TESQUE, catalog number: 32777-44 )
  13. Protease inhibitor cocktail (100x) (NACALAI TESQUE, catalog number: 25955-11 ) (for inhibition of various proteases’ activity such as trypsin, in ddH2O; stored at -20 °C)
  14. NaCl (Wako Pure Chemical Industries, catalog number: 191-01665) (for DT40)
  15. Na2HPO4
  16. KCl
  17. KH2PO4
  18. Tris/HCl (pH 8.0) (Sigma-Aldrich, catalog number: T6791 ) (Stored at room temperature)
  19. Glycerol
  20. Bromophenol blue (BPB)
  21. Sodium dodecyl sulfate
  22. Nonidet P-40 (NACALAI TESQUE, catalog number: 23640-94 ) (for HCT116)
  23. 20 mM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk) (Promega, catalog number: G7231 ) (for inhibition of PNGase activity)
  24. 10 mM Z-Leu-Leu-Leu-CHO (MG132) (PEPTIDE INSTITUTE, catalog number: 3175-v ) (in DMSO; inhibition of proteasomal activity, stored at -20 °C)
  25. 1 M dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 041-08976 ) (in water; for reduction of proteins)
  26. Anti-β-actin antibody (Wako Pure Chemical Industries, catalog number: 017-24573 )
  27. Anti-myc antibody (MEDICAL & BIOLOGICAL LABORATORIES, catalog number: 562 )
  28. Phosphate buffered saline (PBS) (see Recipes)
  29. 2x sodium dodecyl sulfate (SDS) sample buffer (pH 6.8) (see Recipes)
  30. Buffer A (see Recipes)
  31. Buffer B (see Recipes)

Equipment

  1. High speed refrigerated micro centrifuge (Tomy, model: MX-301 )
  2. mPAGE (ATTO, model: AE-6530 ) (Using hand-made 10% gel)
  3. Transfer equipment (ATTO, model: WSE-4020 )
  4. Heat block (TAITEC, model: DTU-1BN )
  5. Micro porator (Digital Bio, model: MP-100 )

Software

  1. ImageJ

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Ninagawa, S. and Mori, K. (2016). Trypsin Sensitivity Assay to Study the Folding Status of Proteins. Bio-protocol 6(19): e1953. DOI: 10.21769/BioProtoc.1953.
  2. Ninagawa, S., Okada, T., Sumitomo, Y., Horimoto, S., Sugimoto, T., Ishikawa, T., Takeda, S., Yamamoto, T., Suzuki, T., Kamiya, Y., Kato, K. and Mori, K. (2015). Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway. J Cell Biol 211(4): 775-784.

    3. ris ris Download Citation in RIS Format

分类

生物化学 > 蛋白质 > 修饰

生物化学 > 蛋白质 > 结构

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件