Detection of phosphorylated T and B cell antigen receptor species by Phos-tag SDS- and Blue Native-PAGE.
Immunol Lett
; 130(1-2): 51-6, 2010 May 04.
Article
em En
| MEDLINE
| ID: mdl-20005898
ABSTRACT
Detection of phospho-proteins and differently phosphorylated forms of the same protein are important in understanding cell behaviour. One novel method is Phos-tag SDS-PAGE. A dinuclear Mn(2+) complex that binds to phosphate groups (the Phos-tag) is covalently attached to the polyacrylamide gel matrix. Thus, phosphorylated proteins are retarded in their migration and can be distinguished from their non-phosphorylated counterparts. We applied Phos-tag SDS-PAGE to the analysis of the zeta, CD3epsilon and CD3delta subunits of the T cell antigen receptor (TCR-CD3). Pervanadate stimulation generated six different phospho-zeta and each two different CD3epsilon and CD3delta forms. This corresponds to the phosphorylatable tyrosines on their cytoplasmic tails. The phosphorylation pattern was compatible with random phosphorylation events. Further, we showed that the Phos-tag technology can be applied to Blue Native (BN)-PAGE. This extends the applicability to the analysis of native protein complexes. Upon pervanadate stimulation the TCR-CD3 complex was predominantly detected as two distinct phospho-complexes. In contrast, the B cell antigen receptor (BCR) appeared as one phospho-form. Thus, Phos-tag BN-PAGE is useful for the analysis of different phosphorylation states of multiprotein complexes.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Receptores de Antígenos de Linfócitos B
/
Complexo Receptor-CD3 de Antígeno de Linfócitos T
/
Complexos Multiproteicos
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Immunol Lett
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Alemanha