RESUMO
The activation of class I phosphoinositide 3-kinases (PI(3)Ks) by cell surface receptors represents the initiation of a large and complex signaling network that couples many growth factors, antigens, and inflammatory stimuli to important cellular responses, such as cell growth, survival, and movement. The most direct measurement of class I PI(3)K activity in cells is the rate of production of its lipid product, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. This chapter describes in detail two approaches used to estimate the levels of PtdIns(3,4,5)P(3) in cells. One approach uses radiotracer labeling of cells, lipid extraction, deacylation, and subsequent quantitation of phosphoinositides by anion-exchange high-performance liquid chromatography. The second approach uses a novel, nonradioactive assay in which the cellular lipids are extracted, phosphoinositides are enriched through binding to a neomycin matrix, dried onto a nitrocellulose membrane, and PtdIns(3,4,5)P(3) quantified by a protein-lipid overlay approach using a GRP(1) PH domain probe.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/análise , 1-Fosfatidilinositol 4-Quinase/isolamento & purificação , Animais , Linhagem Celular , Fenômenos Fisiológicos Celulares , Cromatografia por Troca Iônica/métodos , Humanos , Indicadores e Reagentes , Marcação por Isótopo/métodos , Lipídeos/isolamento & purificação , Neutrófilos/fisiologia , Fosfatos/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/isolamento & purificação , Fosfatidilinositóis/metabolismo , Radioisótopos de Fósforo/isolamento & purificação , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/isolamento & purificaçãoRESUMO
This article presents a simple and rapid method for removal of unincorporated label and proteins from DNA sequencing reactions by using Wizard purification resin. This method can be successfully applied for preparation of end-labeled oligonucleotides free of unincorporated label, which is important in experiments (including DNA sequencing) when the level of background should be as low as possible. Also, this method is effective in removal of proteins from DNA sequencing reactions.