放射性脉冲追踪分析和免疫沉淀法

Javier G.  Magadán

doi:10.21769/BioProtoc.1101

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

Radioactive Pulse-Chase Analysis and Immunoprecipitation

放射性脉冲追踪分析和免疫沉淀法

Javier G. Magadán email

发布: 2014年04月20日第4卷第8期 DOI: 10.21769/BioProtoc.1101 浏览次数: 13230

评审: Anonymous reviewer(s)

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Journal of Virology, featuring study using the protocol.

Sep 2013

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

嗜肺军团菌效应蛋白SidJ体外谷氨酰化活性的放射性测定

Alan G. Sulpizio [...] Yuxin Mao

2020年10月05日 4021 阅读

基于荧光读数的漂浮分析法研究肠道病毒周边膜蛋白2C的膜结合特性

Kasturika Shankar [...] Lars-Anders Carlson

2025年04月05日 1909 阅读

利用酰基树脂辅助捕获法（Acyl-RAC）检测与分析S-酰化蛋白

Dina A. Abdulrahman and Michael Veit

2025年04月05日 2237 阅读

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

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

Equipment

Note: Designated for working with radioactive materials.

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