水解蛋白片段的分离和分析

Jun Ni

doi:10.21769/BioProtoc.232

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Peer-reviewed

Proteolytic Fragment Isolation and Analysis (ex. N-terminal GST-tagged CLAVATA3 Protein GST-CLV3)

水解蛋白片段的分离和分析

JN Jun Ni

发布: 2012年07月20日第2卷第14期 DOI: 10.21769/BioProtoc.232 浏览次数: 12257

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Abstract

It has become clear that the post-embryonic growth and development of plants requires properly controlled short distance cell-to-cell communication not only through the historically well-known phytohormones, but also through secreted small peptide signals. This protocol demonstrates an example of how to isolate small peptides (< 10 daltons) from complex protein mixtures (e.g. cauliflower meristem protein extraction) for MS/MS analysis.

Keywords: CLAVATA3 (CLAVATA3)

Meristem protein isolation (分生组织蛋白质分离)

Proteolytic processing (蛋白水解处理)

Background

Materials and Reagents

Glutathione Sepharose 4B (Amersham biosciences , catalog number: 17-0756-01 )
Glutathione (Thermo Fisher Scientific, catalog number: 78259 )
Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
Triton X-100 (Pierce, catalog number: 85111 )
GST-mCLV3 proteins
Tris-HCl
Hepes (Sigma-Aldrich, catalog number: H3375 )
EDTA (Sigma-Aldrich, catalog nummber: ED100g )
Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number: 78830-1G )
Aprotinin (Sigma-Aldrich, catalog number: A4529-1MG )
Chymostatin (Sigma-Aldrich, catalog number: C7268-1MG )
Leupeptin (Sigma-Aldrich, catalog number: L2884-5MG )
Eluting buffer (see Recipes)
Cauliflower extraction buffer (see Recipes)

Equipment

Rotor
Microcon YM-10 centrifugal filter (EMD Millipore)
Tabletop centrifuge
Mass spectrometer (4700 proteomics analyzer)

Procedure

English

中文翻译

文章信息

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如何引用

Ni, J. (2012). Proteolytic Fragment Isolation and Analysis (ex. N-terminal GST-tagged CLAVATA3 Protein GST-CLV3). Bio-protocol 2(14): e232. DOI: 10.21769/BioProtoc.232.