Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates

被引:21
作者
AlShami, A
Gilbert, C
Barabe, F
Gaudry, M
Naccache, PH
机构
[1] CHUL,CTR RECH,CTR RECH RHEUMATOL & IMMUNOL,ST FOY,PQ G1V 4G2,CANADA
[2] UNIV LAVAL,DEPT MED,ST FOY,PQ G1V 4G2,CANADA
[3] LAB IMMUNOL & HEMATOL,F-75877 PARIS 18,FRANCE
关键词
neutrophil; tyrosine phosphorylation; Cbl; Lyn; immunoblotting; immunoprecipitation;
D O I
10.1016/S0022-1759(97)00004-5
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Activation of various cell types by different agonists is known to stimulate a transient increase in the level of tyrosine phosphorylation of certain cellular proteins. Such phosphorylation is essential for mediating signalling by these agonists. The preservation of the tyrosine phosphorylation of proteins in lysates has proven to be a difficult task in neutrophils because of their large arsenal of proteases and phosphatases. Here we describe a technique that we found useful for preserving the tyrosine phosphorylation of cellular proteins. The technique depends on the denaturing lysis of neutrophils followed by the removal of the denaturing agents using Sephadex columns. Preparing neutrophil lysates by this technique has proven to be reliable in terms of maintaining the stability of the tyrosine phosphorylated proteins of various molecular weights and their subsequent immunoprecipitation and identification.
引用
收藏
页码:183 / 191
页数:9
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