Enhanced chemiluminescence protein labeling

FIGU R E 7 A similar set of substrates is tyrosine phosphorylated during activation of either intact taenia coli, cultured VSMC, or staphylococcal a-toxin-permeabilized deal longitudinal smooth muscle. In these experiments, tyrosine.-phosphorylated substrates were detected by immunoblotting with antiphosphotyrosine antibodies and enhanced chemiluminescence technology rather than the less sensitive I-labeled protein A technology used in Fig. 2. Stimulation of guinea pig taenia coli with either 10 jcM carbachol (Carb) or 1.5 mM vanadate (Van) resulted in pronounced tyrosine phosphorylation of at least nine substrates with apparent masses of 42-45, 50, 70, 80-85, 95,100, 110, 116, and 205 kDa. In like fashion, stimulation of canine femoral VSMC with 100 jjlM phenylephrine (PE) resulted in enhanced tyrosine phosphorylation of a similar set of substrates (however, note that qualitative differences were evident with respect to some substrates, such as the one of 205 kDa). Similarly, the same substrates appeared to be tyrosine phosphorylated when permeabilized ileal smooth muscle was contracted with Ca + (pCa 4.5). From Di Salvo et al. (1994), Fig. 5, p. 1438. 

Microarray detection methods In the early days of protein microarray analysis, mostly radioisotope-based labeling was applied, especially for phosphorylated proteins [99]. Later, stable isotope-coded amino acids (SILAC) proved useful in in vivo incorporation to cell cultures [100]. Both methods were very sensitive, requiring only minute sample amounts to reveal protein expression differences [101]. The use of an enhanced chemiluminescence (ECL) technique using X-ray film exposure or a phosphor imager instrument was. 

---