RESUMEN
A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.
Asunto(s)
Focalización Isoeléctrica/métodos , Proteínas/metabolismo , Transducción de Señal , Línea Celular Tumoral , Humanos , Punto Isoeléctrico , Fosforilación , Isoformas de Proteínas/metabolismo , Sensibilidad y EspecificidadRESUMEN
Previous transfection studies of cationic peptoid polymers (N-substituted polyglycines) and cationic lipitoid polymers (peptoid-phospholipid conjugates) have shown that only the polymers which possessed a repeating (cationic, hydrophobic, hydrophobic) substituent sequence are efficient in gene transfer in vitro. To determine if there is a physical attribute of peptoid and lipitoid complexes that correlates with efficient gene transfection, biophysical, and transfection measurements were performed with polymer:DNA complexes containing each of seven structurally diverse peptoid polymers and two lipitoids that possess different hydrophobic substituents. These measurements revealed that the biophysical properties of these complexes (size, zeta-potential, ethidium bromide exclusion) varied with polymer structure and complex (+/-) charge ratio but were not directly predictive of transfection efficiency. Unique alterations in the circular dichroism spectra of DNA were observed in complexes containing several of the peptoids and both lipitoids, although FTIR spectroscopy demonstrated that the DNA remained in the B-form. The lack of correlations between the physical properties and the transfection activities of these polyplexes suggests that a further subpopulation examination of these complexes by these methods may reveal hidden structure-activity relationships.